Methods and compositions for administration of trpv1 agonists

ABSTRACT

Compositions are provided that contain a TRPV1 agonist, such as capsaicin, and a solvent system. Topical application of the composition results in rapid delivery of agonist to the dermis and epidermis. Method of using the compositions for reducing nociceptive nerve fiber function in subjects, and for treatment of capsaicin-responsive conditions are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. provisional patent applicationNos. 60/462,457 and 60/462,040, both filed Apr. 10, 2003, and60/499,062, filed Aug. 29, 2003. The contents of these applications areincorporated by reference in their entirety and for all purposes.

FIELD OF THE INVENTION

The invention provides compositions and methods for reducing the densityof sensory nerve fibers in tissue and amelioration ofcapsaicin-responsive conditions, and finds application in the field ofmedicine.

BACKGROUND

The transient receptor potential vanilloid-1 (TRPV1) is acapsaicin-responsive ligand-gated cation channel selectively expressedon small, unmyelinated peripheral nerve fibers (cutaneous nociceptors).See Caterina and Julius, 2001, “The vanilloid receptor: a moleculargateway to the pain pathway,” Annu Rev Neurosci. 24:487-517; and Montellet al., 2002, “A unified nomenclature for the superfamily of TRP cationchannels,” Mol. Cell. 9:229-31. When TRPV1 is activated by agonists suchas capsaicin and other factors such as heat and acidosis, calcium entersthe cell and pain signals are initiated. After disease or injury,cutaneous nociceptors may become persistently hyperactive, spontaneouslytransmitting excessive pain signals to the spinal cord in the absence ofpainful stimuli, resulting in various types of chronic pain. When TRPV1is continuously activated through prolonged exposure to an agonist(e.g., capsaicin), excessive calcium enters the nerve fiber, initiatingprocesses that result in long-term yet reversible impairment ofnociceptor function. This is believed to be the mechanism by whichapplication of capsaicin provides relief from pain.

Capsaicin may be effective for amelioration of conditions or diseasesother than pain, as well. For example, capsaicin acts as ananti-inflammatory agent, counter-irritant, antipruritic, anti-psoriaticand anti-itch agent (for review, see Szallasi and Blumberg, 1999,“Vanilloid (Capsaicin) Receptors and Mechanisms,” Pharm Revs,51:159-211). In addition, capsaicin has been reported to cause apoptosisand/or inhibit proliferation of malignant cancer cells (for review, seeSurh, 2002, “More Than Spice: Capsaicin in Hot Chili Peppers Makes TumorCells Commit Suicide,” J Nat Cancer Inst, 94:1263-65), and to reducesinonasal polyps (Baudoin et al., 2000, “Capsaicin significantly reducessinonasal polyps” Acta Otolaryngol 120:307-11).

Low-concentration capsaicin creams have been used for years to treatpainful neuropathies and musculoskeletal pain, but their use is limitedbecause they are painful and inconvenient to apply, normally requiringmultiple daily applications for only modest relief. Recently, a highconcentration capsaicin patch has been developed (NGX-4010; NeurogesX,Inc.) that is believed to provide effective and sustained relief frompain.

The present invention provides additional methods and compositions foradministration of capsaicin and other TRPV1 agonists.

BRIEF SUMMARY OF THE INVENTION

The invention relates to methods, compositions, and devices foradministration of TRPV1 agonists, such as capsaicin, to individuals inneed of treatment.

In one aspect, the invention provides a method of reducing the densityof functional nociceptive nerve fibers in a selected region of a subjectby contacting the region with a composition that contains a TRPV1agonist and a solvent system by one or more penetration enhancers, wheresaid composition delivers at least about 3 nmoles agonist to skin asmeasured in a mouse skin absorption assay. In one embodiment, thecomposition is an immediate-release composition. In one embodiment, thecontacting is under nonocclusive conditions. In one embodiment, thecontacting is under nonadherent conditions. In one embodiment, at leastabout 5 μL of the composition is delivered to each cm² of the region inabout 15 minutes. In an embodiment, a 15 minute application of thecomposition to skin of a mammal results in a decrease in the density offunctional nociceptive nerve fibers by at least about 20%. In anembodiment, the density of functional nociceptive nerve fibers isdecreased by at least about 50%. In an embodiment the mammal is a mouse.In an embodiment, the mammal is a human.

In one aspect, the invention provides a method of treating acapsaicin-responsive condition in a subject by administration of acomposition that contains a TRPV1 agonist and at least one penetrationenhancer, where said composition delivers at least about 3 nmolesagonist to skin as measured in a mouse skin absorption assay. In anembodiment, the composition is an immediate release composition. In anembodiment, the administration is non-occlusive and/or non-adherent. Inone embodiment, the capsaicin-responsive condition is neuropathic pain,pain produced by mixed nociceptive and neuropathic etiologies,inflammatory hyperalgesia, vulvodynia, interstitial cystitis, overactivebladder, prostatic hyperplasia, rhinitis, rectal hypersensitivity,burning mouth syndrome, oral mucositis, herpes, prostatic hypertrophy,dermatitis, pruritis, itch, tinnitus, psoriasis, warts, skin cancers,headaches, or wrinkles. In some embodiments, the composition is appliedto an area on the surface of skin or mucosa.

In a related aspect, the invention provides a method of treating acapsaicin-responsive condition in a subject, by administration of acomposition that contains a TRPV1 agonist and at least two penetrationenhancers, where said composition delivers at least about 3 nmolesagonist to skin as measured in a mouse skin absorption assay.

In various embodiments, the composition delivers at least about 6 nmolesagonist to skin, at least about 16 nmoles, at least about 32 nmoles, atleast about 49 nmoles, or at least about 65 nmoles agonist to skin in amouse skin absorption assay.

In an embodiment, the composition has a depot effect of less than about0.25 as measured in a mouse skin absorption assay. In some embodimentsthe depot effect is less than about 0.1, less than about 0.02, or lessthan about 0.001.

In an embodiment, the composition contains the TRPV1 agonist and asolvent system, where penetration enhancer(s) make up at least 20% (v/v)of the solvent system. In other embodiments, penetration enhancer(s)make up at least 50% (v/v), at least 90%, at least 95% or substantiallyall of the solvent system. In an embodiment, the composition comprisesthe TRPV1 agonist (e.g., a vanilloid such as capsaicin) at aconcentration of from 0.05% (w/v) to 60% (w/v).

In an embodiment the solvent system contains a penetration enhancerselected that is an ether, ester, alcohol, fatty acid, fatty acid ester,fatty alcohol, polyol, terpene, or amine. In one embodiment, the solventsystem contains a penetration enhancer selected from 1-menthone,dimethyl isosorbide, caprylic alcohol, lauryl alcohol, oleyl alcohol,ethylene glycol, diethylene glycol, propylene glycol, triethyleneglycol, butylene glycol, valeric acid, pelargonic acid, caproic acid,caprylic acid, lauric acid, oleic acid, isovaleric acid, isopropylbutyrate, isopropyl hexanoate, butyl acetate, methyl acetate, methylvalerate, ethyl oleate, poloxamer, d-piperitone, methylnonenoic acid,methylnonenoic alcohol, and d-pulegone.

The invention provides a pharmaceutical composition containing atherapeutically effective amount of a TRPV1 agonist and one or morepenetration enhancers, and optionally one or more additionaltherapeutically active agents, where said composition delivers at leastabout 3 nmoles agonist to skin as measured in a mouse skin absorptionassay. In an embodiment, the pharmaceutical composition is in a formsuitable for administration to a subject. In an embodiment, theconcentration of capsaicin is greater than 0.05% and less than 20%.

In one embodiment, the composition that comprises a TRPV1 agonist, andoptionally one or more additional therapeutically active agents, in asolvent system by one or more penetration enhancers, where said one ormore penetration enhancers, taken together, constitute at least about50% (v/v), and up to 100%, of the solvent system. In an embodiment, thecomposition contains another therapeutically active agent, such as alocal anesthetic.

In another aspect, the invention provides a system for treating acapsaicin-responsive condition, the system containing the TRPV1 agonistcomposition or microemulsion and a non-occlusive, non-adherentapplicator device for applying the formulation to skin or a mucosalsurface. In an embodiment, the applicator device is pre-filled with thecomposition. Alternatively, the composition is contained in a containerseparate from the device. In a related embodiment, a kit containing thecomposition or system and a cleaning composition for removal of agonistis provided.

In another aspect, the invention provides a microemulsion containing aTRPV1 agonist such as capsaicin, as well as methods of treatment usingthe microemulsion.

In another aspect, the invention provides a method for ranking two ormore compositions according to their utility for therapeutic delivery ofa TRPV1 agonist to a subject by determining for each composition thedepot effect for a solution consisting of the composition and the TRPV1agonist or a different TRPV1 agonist, comparing the values obtained foreach composition, and ranking them compositions according to the values,where a composition with a lower value is ranked more suitable fortherapeutic delivery of the TRPV1 agonist.

In another aspect, the invention provides a method of increasing theamount of a topically applied molecule that enters the epidermal anddermal layers by topically applying the molecule in a compositioncontaining methylnonenoyl alcohol or methylnonenoic acid. In a relatedanother aspect, the invention provides a pharmaceutical compositioncontaining capsaicin and methylnonenoyl alcohol or methylnonenoic acid.

In another aspect, the invention provides a method of delivering a TRPV1agonist to the epidermis and dermis underlying a 1 cm² area of a skin ormucosal surface of a mammal by contacting the area with a compositioncomprising the TRPV1 agonist and at least one penetration enhancer,where 15 or 30 minutes after contacting at least about 3 nmole of theTRPV1 agonist is retained in the epidermis and dermis. In certainembodiments, the density of functional nociceptive nerve fibers in theepidermis and dermis is decreased by at least about 20% when measuredafter the contacting step.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows reduction of nerve fiber density in skin (nude mouse) afteradministration of a TRPV1 agonist.

FIG. 2 shows reduction of nerve fiber density in vulva (rat) afteradministration of TRPV1 agonists. Transcutol® is diethylene glycolmonoethyl ether (DGME).

FIG. 3 shows pain behavior after application of TRPV1 agonist to vulva(rat).

FIG. 4 shows pain behavior after application of TRPV1 agonist to skin(rat dorsal paw).

DETAILED DESCRIPTION 1. Introduction

The present inventions relate, in part, to the discovery thatadministration of TRPV1 agonist under conditions in which a significantamount of agonist is rapidly and efficiently delivered to, and retainedin, the skin provides surprising benefits. In particular, such deliveryresults in significant reduction in the density of functional cutaneousor mucosal nociceptor nerve fibers in a treated area following only abrief exposure to the agonist (see, e.g., Examples 1, 2 and 3, infra).Further, it is believed that the discomfort ordinarily associated withcontact with TRPV1 agonists such as capsaicin is reduced when agonist israpidly and efficiently delivered to, and retained in, the skin ormucosa (see, e.g., Examples 4 and 5, infra).

In a related aspect, the invention provides a method of treating acapsaicin-responsive condition in a subject by topical administration ofa composition containing capsaicin or other TRPV1 agonist underconditions in which a significant amount of agonist is rapidly andefficiently delivered to, and retained in, the skin or mucosa.

In one aspect of the invention, a composition containing a TRPV1agonist, optionally containing additional therapeutically activeagent(s), containing a solvent system containing at least onepenetration enhancer, and optionally containing other components asdescribed below, is contacted with the target region of the subject'sbody (e.g., skin or mucosa). For clarity, this composition is sometimesreferred to as the “administered composition.” In some embodiments, thesolvent system is characterized in that one or more penetrationenhancers make up a high proportion of the solvent system.

2. Definitions and Conventions

The following definitions are provided to assist the reader inunderstanding the invention. Unless otherwise defined, all terms of art,notations and other scientific or medical terms or terminology usedherein are intended to have the meanings commonly understood by those ofskill in the chemical and medical arts. In some cases, terms withcommonly understood meanings are defined herein for clarity and/or forready reference, and the inclusion of such definitions herein should notnecessarily be construed to represent a substantial difference over thedefinition of the term as generally understood in the art.

“Therapeutically effective amount” or “Therapeutically effective dose”refers to the quantity or dose of an agent required to produce aclinically desired result such as a biological or chemical response(e.g., a reduction in density of functional cutaneous or mucosalnociceptor nerve fibers in a subject in need of such reduction),alleviation or amelioration of one or more symptoms of a disease orcondition, diminishment of extent of disease, or stabilized state ofdisease.

“Treating” a condition or patient refers to taking steps to obtainbeneficial or desired results, including clinical results. For purposesof this invention, beneficial or desired clinical results include, butare not limited to, alleviation or amelioration of one or more symptomsof diminishment of extent of disease, delay or slowing of diseaseprogression, amelioration, palliation or stabilization of the diseasestate, and other beneficial results described below.

“Pharmaceutically acceptable salt” refers to an acidic or basic saltthat is toxicologically safe for administration to a subject, includingwithout limitation, phosphate, sulfate, lactate, napsylate, mesylate,hydrochloride, sodium, potassium, n-methylglucamine, and tromethaminesalts.

“Local administration,” “topical administration,” “topically,” andgrammatical equivalents, refer to administration of a biologicallyactive compound to a pre-defined or definite area of the body, such asto a defined or limited area of the skin surface, mucous membrane, aspecified organ, a specified appendage or region (e.g., foot). Local ortopical administration, as used herein, does not include administrationby subdermal injection.

“Fully soluble” or “completely dissolved” or “fully in solution” refersto a visually clear homogenous solution with substantially no suspendedor undissolved particles of therapeutically active agents. Aquantitative determination of solution clarity can be made bymeasurements of turbidimetry, e.g. the reduction of transparency of aliquid caused by the presence of undissolved matter (see Lawler, 1995,Turbidimetry and Nephelometry Encyclopedia of Analytical Science, ed. P.Worsfold, Academic Press Ltd, UK). Instruments such as the Model 2100ANor 2100N turbidimeter (Hach Co., Loveland, Colo.) may be used. Usuallythe turbidity of a composition containing a therapeutically active agentis less than about 10 NTU (nephelometric turbidity unit), more usuallyless than about 5 NTU or less than about 3 NTU.

“Stratum corneum” refers to the outer layer of the skin that is theprimary barrier layer. The stratum corneum creates the rate-limitingbarrier for diffusion of an active agent across the skin.

“Penetration enhancer” refers to an agent that improves the rate ofpercutaneous transport of an active agent across the skin for use anddelivery of active agents to organisms such as mammals.

An “individual” or “subject” is a vertebrate, preferably a mammal, andoften a human. Mammals include, but are not limited to, humans,non-human primates, experimental model animals (e.g., mice and rats),agriculturally important animals, and pets.

As used herein, manufactured or formulated “under GMP standards,” whenreferring to a pharmaceutical composition means the composition is infull compliance with all Good Manufacturing Practice (GMP) regulationsof the U.S. Food and Drug Administration.

“Nerve fiber functionality (NFF)” is a measure of the functional orstructural inactivation of nociceptive (TRPV1-expressing) sensory nervefibers. A change in NFF can be expressed as a change in the density offunctional nerve fibers identified by immunostaining and morphology, asdescribed below. Alternatively, a change in NFF can be expressed as achange in a sensitivity of the nerve fiber (e.g., to changes intemperature).

As used herein, the terms “deliver,” “delivering” and grammaticalequivalents (e.g., as in “delivery of agonist to epidermis and dermis”)refers to taking action that results in transfer of an agent (e.g.,TRPV1 agonist) to a target tissue (e.g., epidermis and dermis). Forexample, capsaicin can be delivered to dermis by applying acapsaicin-containing composition of the invention to the unbrokensurface of skin overlying the dermis.

As used herein, the term “retained” (e.g., as in “agonist retained inskin”) refers to the amount of an agent found in a specified tissue(e.g., epidermis and dermis) at a specified point in time (e.g., 15minutes after application of an agonist-containing composition).

The following conventions and abbreviations are used in the descriptionbelow: Unless otherwise indicated, temperatures are in degreescentigrade and all measurements are made at 1 atmosphere and at atemperature of 23° C. to 32° C. Abbreviations used in this disclosureinclude “μL” (microliter); “mL” (milliliter); “nmole” (nanomole); “PE”(penetration enhancer); “TAA” (therapeutically active agent);weight/volume (w/v); volume/volume (v/v). References to a specified timeafter administration of a TRPV1 agonist-containing composition (e.g.,“15 minutes”) refer the the time beginning with the initial firstcontact of the composition with the subject (e.g., the time ofapplication). If not otherwise specified or apparent from context,assays can be conducted 15 or 30 minutes after administering acomposition, and values normalized to administration to a 1 cm² area.

3. TRPV1 Agonists

TRPV1 agonists useful in the present invention include capsaicin,capsaicin analogs and derivatives, and other low molecular weightcompounds (i.e., MW<1000) that agonize the TRPV1. Capsaicin can beconsidered the prototypical TRPV1 agonist. Capsaicin (also called8-methyl-N-vanillyl-trans-6-nonenamide;(6E)-N-[(4-hydroxy-3-methoxyphenyl) methyl]-8-methylnon-6-enamide;N-[(4-hydroxy-3-methoxyphenyl) methyl]-8-methyl-(6E)-6-nonenamide;N-(3-methoxy-4-hydroxybenzyl)-8-methylnon tran-6-enamide;(E)-N-[(4-hydroxy-3-methoxyphenyl)methyl]-8-methyl-6-nonenamide) has thefollowing chemical structure:

In addition to capsaicin, a variety of capsaicin analogs andderivatives, and other TRPV1 agonists may be administered. Vanilloids,such as capsaicinoids, are an example of useful TRPV1 agonists.Exemplary vanilloids for use according to the invention includeN-vanillyl-alkanedienamides, N-vanillyl-alkanedienyls,N-vanillyl-cis-monounsaturated alkenamides, capsaicin, dihydrocapsaicin,norhydrocapsaicin, nordihydrocapsaicin, homocapsaicin, andhomodihydrocapsaicin.

In another embodiment, the TRPV1 agonist is a compound lacking thevanillyl function, such as piperine or a dialdehyde sesquiterpene (forexample warburganal, polygodial, or isovelleral). In another embodiment,the TRPV1 agonist is a triprenyl phenol, such as scutigeral. Additionalexemplary TRPV1 agonists are described in U.S. Pat. Nos. 4,599,342;5,962,532; 5,762,963; 5,221,692; 4,313,958; 4,532,139; 4,544,668;4,564,633; 4,544,669; 4,493,848; 4,532,139; 4,564,633; and 4,544,668;and PCT publication WO 00/50387. Other useful TRPV1 agonists includepharmacologically active gingerols, piperines, shogaols, and morespecifically guaiacol, eugenol, zingerone, civamide, nonivamide,nuvanil, olvanil, NE-19550, NE-21610, and NE-28345 (see Dray et al.,1990, Eur. J. Pharmacol 181:289-93 and Brand et al., 1990, AgentsActions 31:329-40), resiniferatoxin, resiniferatoxin analogs, andresiniferatoxin derivatives (e.g., tinyatoxin). Any active geometric- orstereo-isomer of the forgoing agonists may be used.

Other TRPV1 agonists are vanilloids that have TRVP1 receptor-bindingmoieties such as mono-phenolic mono-substituted benzylamine amidatedwith an aliphatic cyclized, normal or branched substitution. Still otheruseful TRPV1 agonists for practicing the invention can be readilyidentified using standard methodology, such as that described in U.S.patent publication US20030104085. Useful assays for identification ofTRPV1 agonists include, without limitation, receptor binding assays;functional assessments of stimulation of calcium influx or membranepotential in cells expressing the TRPV1 receptor, assays for the abilityto induce cell death in such cells (e.g., selective ablation of C-fiberneurons), and other assays known in the art.

Mixtures of agonists and pharmaceutically acceptable salts of any of theforegoing may also be used. See Szallasi and Blumberg, 1999,Pharmacological Reviews 51:159-211, U.S. Pat. No. 5,879,696, andreferences therein.

4. Rapid and High Quantity Delivery of TRPV1 Agonists

The present invention relates, in part, to the discovery thatadministration of a TRPV1 agonist under conditions in which asignificant amount of agonist is rapidly delivered to, and preferablyretained in, the skin provides surprising benefits.

In one aspect, the invention provides a method for treating acapsaicin-responsive condition by administering a composition thatcontains a TRPV1 agonist and one or more penetration enhancers. In anembodiment, the composition delivers at least about 3 nmoles agonist toskin as measured in a mouse skin absorption assay. The mouse skinabsorption assay is described in detail below. As discussed below,delivery of a specified molar amount of agonist to skin in a mouse skinabsorption assay refers to the amount delivered per 1 cm² (as normalizedfrom a 0.8 cm² area of skin used in the assay) in fifteen (15) minutesunder the assay conditions.

In a related aspect, the invention provides a method of reducing thedensity of functional nociceptive nerve fibers in a selected tissue of asubject by contacting the tissue with a composition that contains aTRPV1 agonist and one or more penetration enhancers. In an embodiment,the composition delivers at least about 3 nmoles agonist to skin asmeasured in a mouse skin absorption assay.

In a related aspect, the invention provides a pharmaceutical compositioncontaining a TRPV1 agonist and one or more penetration enhancers, wherethe composition delivers at least about 3 nmoles agonist to skin asmeasured in a mouse skin absorption assay.

In one embodiment of the invention, the TRPV1 agonist is capsaicin andthe amount of agonist delivered is at least about 3 nmoles, at leastabout 6 nmoles, at least about 9 nmoles, at least about 16 nmoles, atleast about 32 nmoles, at least about 49 nmoles, or at least about 65nmoles as measured in a mouse skin absorption assay. In an embodiment,the TRPV1 agonist is capsaicin and the amount of agonist delivered is inthe range of from about 3 nmoles to about 290 nmoles, such as a rangebounded by a lower limit of 3 nmoles, 6 nmoles, 9 nmoles, 16 nmoles, 32nmoles, or 49 nmoles, and an independently selected upper limit of 6nmoles, 9 nmoles, 16 nmoles, 32 nmoles, 49 nmoles, 65 nmoles, 75 nmoles,90 nmoles, 120 nmoles, 200 nmoles and 290 nmoles, where the upper limitis higher than the lower limit.

In a related embodiment of the invention, the TRPV1 agonist is anagonist other than capsaicin and the amount of agonist delivered asmeasured in a mouse skin absorption assay is at least about 3 nmoles, atleast about 6 nmoles, at least about 9 nmoles, at least about 16 nmoles,at least about 32 nmoles, at least about 49 nmoles, or at least about 65nmoles, or in the range of from about 3 nmoles to about 290 nmoles, suchas a range bounded by a lower limit of 3 nmoles, 6 nmoles, 9 nmoles, 16nmoles, 32 nmoles, or 49 nmoles, and an independently selected upperlimit of 6 nmoles, 9 nmoles, 16 nmoles, 32 nmoles, 49 nmoles, 65 nmoles,75 nmoles, 90 nmoles, 120 nmoles, 200 nmoles and 290 nmoles, where theupper limit is higher than the lower limit.

In a related embodiment of the invention, the TRPV1 agonist is anagonist other than capsaicin and the amount of agonist delivered asmeasured in a mouse skin absorption assay is the equivalent of at leastabout 3 nmoles capsaicin, at least about 6 nmoles capsaicin, at leastabout 9 nmoles capsaicin, at least about 16 nmoles capsaicin, at leastabout 32 nmoles capsaicin, at least about 49 nmoles capsaicin, or atleast about 65 nmoles capsaicin, or in the range of from about 3 nmolesto about 290 nmoles capsaicin, such as a range bounded by a lower limitof 3 nmoles, 6 nmoles, 9 nmoles, 16 nmoles, 32 nmoles, or 49 nmolescapsaicin, and an independently selected upper limit of 6 nmoles, 9nmoles, 16 nmoles, 32 nmoles, 49 nmoles, 65 nmoles, 75 nmoles, 90nmoles, 120 nmoles, 200 nmoles and 290 nmoles capsaicin, where the upperlimit is higher than the lower limit.

A molar amount of a TRPV1 agonist that is equivalent, as the term isused in this context, to a molar amount (e.g., 3 nmoles) of capsaicincan be determined using standard methodology. Since the potency andefficacy of TRPV1 agonists can vary, it is in some cases it is useful,upon determining an optimal delivery dose for capsaicin, to adjust theconcentration or dosage of a TRPV1 agonist other than capsaicin. Thenumber of moles of a non-capsaicin TRPV1 agonist which will produce thesame degree of a biological effect (e.g., reduced nociceptive nervefiber function) produced by 1 mole of capsaicin is referred to as thecapsaicin equivalent (“CE”). The concept of CE is analogous to that of‘morphine equivalents’ used to predict equivalent analgesic dose levelsof various opioid analgesics (see, e.g., Kaiko, 1986, “Controversy inthe management of chronic cancer pain: therapeutic equivalents of IM andPO morphine” J Pain Symptom Manage. 1:42-5; Hoskin et al., 1991, “Opioidagonist-antagonist drugs in acute and chronic pain states” Drugs41:326-44). CE values are derived from both the potency and efficacy ofthe TRPV1 agonist, compared to capsaicin under identical assayconditions. One way to determine the relative potency and efficacy ofTRPV1 agonists is to use a standardized in vitro assay, such as thosebased on Fluorometric Imaging Plate Reader (FLIPR) technology (Sullivanet al., 1999, “Measurement of [Ca2+] using the Fluorometric ImagingPlate Reader (FLIPR)” Methods Mol Biol. 114:125-33). FLIPR assays havebeen used widely to characterize and compare a large number of TRPV1agonists (see, e.g., Smart et al., 2001, “Characterisation using FLIPRof human vanilloid VR1 receptor pharmacology.” Eur J PharmacoL 417:51-8;Witte et al., 2002, “Use of a fluorescent imaging plate reader-basedcalcium assay to assess pharmacological differences between the humanand rat vanilloid receptor” J Biomol Screen. 7:466-475; and Behrendt etal., 2004, “Characterization of the mouse cold-menthol receptor TRPM8and vanilloid receptor type-1 VR1 using a fluorometric imaging platereader (FLIPR) assay” Br J Pharmacol. 141:737-45). One way to determinethe CE value for a non-capsaicin TRPV1 agonist would be to combine thepotency and efficacy of the compound by assigning values of 0.5 to boththe potency and efficacy measured for capsaicin in a FLIPR assay(yielding a combined value of 1.0). Then the potency and efficacy valuesof another TRPV1 agonist is determined, and normalized to the 0.5 ofcapsaicin. The combined normalized value of another TRPV1 agonist iscompared to the 1.0 of capsaicin to provide the approximate number ofnmoles of the non-capsaicin TRPV1 agonist that is expected to producethe same effect as capsaicin on, for example, nerve fiber functionality(NFF) when applied.

In a further aspect of the invention, the composition delivers the TRPV1agonist with a significant depot effect. As used herein, the “depoteffect” refers to the retention of agonist in skin in a mouse skinassay, and is the ratio of the amount of agonist that passes out of skinto the amount retained in skin in the mouse skin absorption assay.

Thus, in an aspect the invention provides a method for treating acapsaicin-responsive condition by administering a composition thatcontains a TRPV1 agonist and one or more penetration enhancers, wherethe composition delivers 3 nmoles or more than 3 nmoles agonist to skinas measured in a mouse skin absorption assay, and where is significantproportion of the agonist is retained in the skin.

In a related aspect, the invention provides a method of reducing thedensity of functional nociceptive nerve fibers in a selected tissue of asubject by contacting the tissue with a composition that contains aTRPV1 agonist and one or more penetration enhancers where thecomposition delivers 3 nmoles or more than 3 nmoles agonist to skin asmeasured in a mouse skin absorption assay, and where a significantproportion of the agonist is retained in the skin.

In some embodiments, for example, the ratio of the amount of agonistthat enters the receptor chamber in the mouse skin absorption assay tothe amount retained in the skin is less than about 0.25, less than about0.2, less than about 0.15, less than about 0.1, less than about 0.04,less than about 0.02, less than about 0.01, less than about 0.004, lessthan about 0.002, less than about 0.0015, or less than about 0.001. Insome embodiments, the ratio of the amount of agonist that enters thereceptor chamber in the mouse skin absorption assay to the amountretained in the skin at a specified time point is between about 0.0001and about 0.25, often between about 0.0001 and about 0.2, sometimesbetween about 0.0001 and about 0.1, and sometimes between about 0.0001and 0.04. In some embodiments, the ratio of the amount of agonist thatenters the receptor chamber in the mouse skin absorption assay to theamount retained in the skin is between about 0.001 and about 0.25, oftenbetween about 0.01 and about 0.2, and sometimes between about 0.1 andabout 0.25.

In one embodiment, the ratio of the amount of agonist in the dermis tothe amount in the epidermis is in the range of 0.5 to 2, as measured ina mouse skin absorption assay. In some embodiments the ratio is in therange of 0.75 to 1.5.

In a related embodiment, the invention provides a method for treating acapsaicin-responsive condition by administering a composition thatcontains a TRPV1 agonist under conditions in which a significant amountof agonist is rapidly delivered to, and preferably retained in, a targettissue such as, but not limited to skin, mucosa, and endothelium). Inanother related embodiment, the invention provides a method for reducingthe density of functional nociceptive nerve fibers in a selected tissueof a subject by contacting the tissue with a composition that contains aTRPV1 agonist under conditions in which a significant amount of agonistis rapidly delivered to, and preferably retained in, the target tissue.In an embodiment, “conditions in which a significant amount of agonistis rapidly delivered to the target tissue,” refers to administration ofa TRPV1 agonist under conditions that result in delivery of at least 3nmoles agonist per cm² surface area of application (e.g., skin or mucosasurface area) within 30 minutes, more often within 15 minutes, andsometimes within 5 minutes.

In one embodiment, conditions in which a significant amount of agonistis rapidly delivered to the skin are defined as those measured in ahuman subject. In one embodiment the human subject is a subject innormal health. In one embodiment, the human subject is a subject in needof treatment for a capsaicin-responsive disease or condition. Thus, inone embodiment, the invention provides a method of reducing the densityof functional nociceptive nerve fibers in a selected tissue region of asubject, by topically administering a significant amount of agonist tothe region in less than about 30 minutes, optionally less than about 15minutes, and optionally in less than about 10 minutes. A significantamount can be at least 3 nmoles, optionally at least 6 nmoles, at leastabout 9 nmoles, at least about 16 nmoles, at least about 32 nmoles, atleast about 49 nmoles, or at least about 65 nmoles, or in the range offrom about 3 nmoles to about 290 nmoles, such as a range bounded by alower limit of 3 nmoles, 6 nmoles, 9 nmoles, 16 moles, 32 nmoles, or 49nmoles, and an independently selected upper limit of 6 nmoles, 9 nmoles,16 nmoles, 32 nmoles, 49 nmoles, 65 nmoles, 75 nmoles, 90 nmoles, 120nmoles, 200 nmoles and 290 nmoles, where the upper limit is higher thanthe lower limit, per cm² of the surface of the region (e.g., skin,mucosa, and endothelium, e.g., bladder).

In an alternative embodiment, conditions in which a significant amountof agonist is rapidly delivered to the skin are defined as thosemeasured in a mouse skin assay.

In one embodiment of the invention, the TRPV1 agonist is capsaicin andthe amount of agonist delivered is at least about 3 nmoles, at leastabout 6 nmoles, at least about 9 nmoles, at least about 16 nmoles, atleast about 32 nmoles, at least about 49 nmoles, or at least about 65nmoles. In an embodiment, the TRPV1 agonist is capsaicin and the amountof agonist delivered is in the range of from about 3 nmoles to about 290nmoles, such as a range bounded by a lower limit of 3 nmoles, 6 nmoles,9 nmoles, 16 nmoles, 32 nmoles, or 49 nmoles, and an independentlyselected upper limit of 6 nmoles, 9 nmoles, 16 nmoles, 32 nmoles, 49nmoles, 65 nmoles, 75 nmoles, 90 nmoles, 120 nmoles, 200 nmoles and 290nmoles, where the upper limit is higher than the lower limit.

In a related embodiment of the invention, the TRPV1 agonist is anagonist other than capsaicin and the amount of agonist delivered is atleast about 3 nmoles, at least about 6 nmoles, at least about 9 nmoles,at least about 16 nmoles, at least about 32 nmoles, at least about 49nmoles, or at least about 65 nmoles, or in the range of from about 3nmoles to about 290 nmoles, such as a range bounded by a lower limit of3 nmoles, 6 nmoles, 9 nmoles, 16 nmoles, 32 nmoles, or 49 nmoles, and anindependently selected upper limit of 6 nmoles, 9 nmoles, 16 nmoles, 32nmoles, 49 nmoles, 65 nmoles, 75 nmoles, 90 nmoles, 120 nmoles, 200nmoles and 290 nmoles, where the upper limit is higher than the lowerlimit.

In a related embodiment of the invention, the TRPV1 agonist is anagonist other than capsaicin and the amount of agonist delivered is theequivalent of at least about 3 nmoles capsaicin, at least about 6 nmolescapsaicin, at least about 9 nmoles capsaicin, at least about 16 nmolescapsaicin, at least about 32 nmoles capsaicin, at least about 49 nmolescapsaicin, or at least about 65 nmoles capsaicin, or in the range offrom about 3 nmoles to about 290 nmoles capsaicin, such as a rangebounded by a lower limit of 3 nmoles, 6 nmoles, 9 nmoles, 16 nmoles, 32nmoles, or 49 nmoles capsaicin, and an independently selected upperlimit of 6 nmoles, 9 nmoles, 16 nmoles, 32 nmoles, 49 nmoles, 65 nmoles,75 nmoles, 90 nmoles, 120 nmoles, 200 nmoles and 290 nmoles capsaicin,where the upper limit is higher than the lower limit.

In a related embodiment, the invention provides a method for treating acapsaicin-responsive condition by administering a composition thatcontains a TRPV1 agonist under conditions in which a significant amountof agonist is delivered to and retained in the tissue. In anotherrelated embodiment, the invention provides a method for reducing thedensity of functional nociceptive nerve fibers in a selected tissue of asubject by contacting the tissue with a composition that contains aTRPV1 agonist under conditions in which a significant amount of agonistis delivered to and retained in skin. Systemic exposure to bolus dosesof TRPV1 agonists poses safety risks for patients. This is because thesereceptors are expressed in nerve fibers which regulate thecardiovascular system (and other organ systems), so consequently a rapidactivation of these nerves is expected to produce rapid changes in heartrate and blood pressure (Zahner et al., 2003, “Cardiac vanilloidreceptor 1-expressing afferent nerves and their role in the cardiogenicsympathetic reflex in rats” J Physiol. 551:515-23). Such changes areproblematic for elderly patients and those with pre-existingcardiovascular disease. Accordingly, the surprising discovery that highand rapid exposures of skin, mucous membranes, and other types of tissueto TRPV1 agonists could be attained as described here without anexpectation of significant systemic drug delivery, allows topicalapplication of TRPV1 agonist-containing formulations with relativelyhigh safety margins. In one aspect the present invention provides amethod of delivering a TRPV1 agonist to the epidermis and dermisunderlying a 1 cm² area of a skin or mucosal surface of a mammal, bycontacting the area with a composition comprising the TRPV1 agonist andat least one penetration enhancer, wherein 30 minutes after saidcontacting at least about 3 nmole of the TRPV1 agonist is retained inthe epidermis and dermis. In a related embodiment, the inventionprovides a method of delivering a TRPV1 agonist to the epidermis anddermis underlying a 1 cm² area of a skin or mucosal surface of a mammal,by contacting the area with a composition comprising the TRPV1 agonistand at least one penetration enhancer, wherein 15 minutes after saidcontacting at least about 3 nmole of the TRPV1 agonist is retained inthe epidermis and dermis. In various embodiments, the amount of TRPV1agonist retained in the epidermis and dermis is at least about 3 nmoles,at least about 6 nmoles, at least about 9 nmoles, at least about 16nmoles, at least about 32 nmoles, at least about 49 nmoles, or at leastabout 65 nmoles. In an embodiment, the TRPV1 agonist is capsaicin andthe amount of agonist delivered is in the range of from about 3 nmolesto about 290 nmoles, such as a range bounded by a lower limit of 3nmoles, 6 nmoles, 9 nmoles, 16 nmoles, 32 nmoles, or 49 nmoles, and anindependently selected upper limit of 6 nmoles, 9 nmoles, 16 nmoles, 32nmoles, 49 nmoles, 65 nmoles, 75 nmoles, 90 nmoles, 120 nmoles, 200nmoles and 290 nmoles, where the upper limit is higher than the lowerlimit.

It will be understood that when measuring the agonist content ofepidermis and dermis underlying a 1 cm² area, the actual cross sectionof tissue assayed can be less than (e.g., 0.8 cm²) or greater than 1cm², and the measured agonist content can be normalized to an amount percm².

In one embodiment, the agonist is contacted with the skin, mucosal orbladder surface in vitro (e.g., using a mouse skin absorption assay orsimilar assay). In another embodiment, the agonist is contacted with theskin, mucosal or bladder surface in vivo (e.g., by applying thecomposition to skin of a human or animal, such as mouse) and a tissuesample (e.g., of the skin surface and underlying dermis and epidermis)is obtained and agonist content determined. Tissue samples can beobtained using routine methods, such as punch biopsy or excision.Agonist content can be determined using quantiative methods such asHPLC-MS (see, Examples), as appropriate for the particular agonist used.Optionally, separate determinations can be made for the dermal andepidermal layers and the values combined.

In one embodiment the TRPV1 agonist is capsaicin.

In an embodiment, the mammal is a human subject. The human subject maybe in normal health, or may suffer from a capsaicin-responsivecondition.

In a further aspect, the amount of agonist delivered to the underlyingtissue (dermis and epidermis) is sufficient to reduce the density offunctional nociceptive nerve fibers in the epidermis and dermis (i.e.,reduced nerve fiber function) by at least about 20% when measured 1, 2,3, 4, 5, 6 or 7 days after said contacting step. In alternativeembodiments the reduction is at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, or at leastabout 80% compared to an untreated region. It will be clear that whenagonist concentration and NFF are determined in the same individualsubject, the determinations are made using different tissues areas. Moreoften, however, assays can be carried out in different subjects toestablish that specified conditions of contacting (using a TRPV1 agonistin combination with compositions of the invention result in thespecified delivery of agonist and/or reduction in NFF.

4.1 the “Mouse Skin Absorption Assay”

The “mouse skin absorption assay” is an in vitro Franz cell-based assayin which skin of Nu/Nu (“nude”) mice is used to determine (1) the amountof agonist that enters the skin following administration of agonist to a0.8 cm² area of skin surface for 15 minutes; (2) the proportion ofagonist in the skin that is found in each of the epidermal and dermallayers; and (3) the amount of agonist that penetrates the skin (i.e.,enters the receptor chamber of the Franz cell). This assay, which isdescribed in detail in Example 1, infra, measures the amount of agonistthat is retained in the dermis and epidermis fifteen (15) minutes afterthe surface of the skin is contacted with a composition containing theagonist. Consistent with reports that in vitro studies with nude mouseskin are predictive of the result obtained in living animals (Venter etal., 2001, “A comparative study of an in situ adapted diffusion cell andan in vitro Franz diffusion cell method for transdermal absorption ofdoxylamine” Eur J Pharm Sci. 13:169-77) delivery of TRPV1 agonist toskin in the mouse skin model correlates with reduction of nerve fiberfunctionality in vivo following administration of the agonist (seeExamples, illustrating a relationship between delivery of a TRPV1agonist into nude mouse skin in vitro and the pharmacological effects oncutaneous nerve fiber immunostaining in in vivo assays). In mammals,physical processes such as diffusion, partitioning and physical bindingvary in a predictable manner (Franz et al., 1992, In: Treatise onControlled Drug Delivery. Edited by A Kydonieus. Marcel Dekker, Inc. NewYork), and in vitro nude mouse skin studies are considered predictive ofthe penetration rates of drug substance and solvents in human skin(Durrheim et al., 1980, “Permeation of hairless mouse skin I:Experimental methods and comparison with human epidermal permeation byalkanols” J. Pharm. Sci. 69:781-6; see also Tojo, 1987, “Mathematicalmodeling of skin permeation of drugs” J. Chem. Eng. Jpn. 20:300-308; andTojo, 1988, “Concentration profile in plasma after transdermal drugdelivery” Int. J. Pharm. 43:201-205). Notably, the low density of hairfollicles in hairless animal species such as the nude mouse brings thesemembranes closer in that respect to human skin (Katz, 1993, “Rationaleand Approaches to Skin Permeation” In: Skin Permeation, Fundamentals andApplication, Edited by J L. Zatz. Allured Publishing Corp. Wheaton,Ill.).

Using the mouse skin absorption assay, several values can be measured.The amounts of agonist that enter the epidermis (“E”) and dermis (“D”)can be measured. (In the values reported in the examples, the amountthat enters the 0.8 cm² cross section of skin is normalized to 1 cm² bymultiplying by 1.25). The ratio of these two values (“E/D”), referred toas the “distribution effect” is a measure of the relative distributionof agonist in the two skin layers, with equal distribution (on either amolar or mass basis, as specified) giving a ratio of 1. The sum of thesetwo values (E plus D) is the total amount of agonist delivered to theskin (“S”) under the assay conditions. The amount of agonist that passesthrough the skin and enters the receptor chamber of the Franz cell (“P”)can also be measured. The ratio of the amount of agonist that passesthrough the skin to the amount that is retained in skin (“P/S”) isreferred to as the “depot effect.” The units of E, D and S can be molar(e.g., nmole agonist) or weight (e.g., microgram agonist). E/D and P/Sare unitless.

5. The Administered Composition

In one aspect of the invention, a TRPV1 agonist is administered as acomposition (“the administered composition”) containing at least onepenetration enhancer. The applied composition of the invention can bedescribed as having three components:

-   -   1. A solvent system in which the TRPV1 agonist is soluble,        containing at least one penetration enhancer;    -   2. The TRPV1 agonist(s) and/or one or more additional        therapeutically active agents;    -   3. Additional components that, if present, account for not more        than 5% (w/v) of the composition.        In some embodiments of the invention the solvent system is        characterized by having a high concentration of penetration        enhancer(s).

5.1 Solvent System

5.1.1 Penetration Enhancers

A solvent system of the invention can contain a penetration enhancer orcombination of penetration enhancers. Penetration enhancers are wellknown in the art, and are compositions that provide marked intradermalor percutaneous delivery of an agent (see Smith and Maibach, inPercutaneous Penetration Enhancers; CRC Press: Florida 1995; pp 1-8,e.g., Table 1; also see Barry, B. W. “Vehicle Effect: What Is anEnhancer?” In: TOPICAL DRUG BIOAVAILABILITY, BIOEQUIVALENCE, ANDPENETRATION. Shah & Maibach. Eds. Plenum Press: New York, 1993; pp261-76).

Without intending to be bound by a specific mechanism, penetrationenhancers are believed to operate by several mechanisms, which include‘shunting’ the drug substance through pores, sweat glands and hairfollicles, and opening the intercellular spaces of the stratum corneum(Asbill et al., 2000, “Enhancement of transdermal drug delivery:chemical and physical approaches,” Crit Rev Ther Drug Carrier Syst.17:621-58). Regarding the latter, the proteinaceous intracellularmatrices of the stratum comeum, together with the diverse biochemicalenvironments of the intercellular domains in the stratum comeum,represent a formidable barrier to drugs before they can reach the deeperparts of epidermis (e.g., the stratum germinativum) and dermis. Onceabsorbed into the stratum comeum, effects of the penetration enhancermay include altering the solvent potential of the stratum comeumbiochemical environment (i.e., the ability of stratum comeum to retaindrug substances in a non-crystalline form), and disordering the orderedstructure of the intercellular lipid region (for example, due toinsertion of the enhancer molecule between the parallel carbon chains ofthe fatty acids). Exemplary penetration enhancers are listed, forillustration and not limitation, below (see, e.g., in Tables 1-3). Otherpenetration enhancers can be identified using routine assays, e.g., invitro skin permeation studies on rat, pig or human skin using Franzdiffusion cells (see Franz et al., “Transdermal Delivery” In: Treatiseon Controlled Drug Delivery. A. Kydonieus. Ed. Marcell Dekker: New York,1992; pp 341-421). Many other methods for evaluation of enhancers areknown in the art, including the high throughput methods of Karande andMitragotri, 2002, “High throughput screening of transdermalformulations” Pharm Res 19:655-60, and Karande and Mitragotri, 2004,“Discovery of transdermal penetration enhancers by high-throughputscreening”).

Penetration enhancers suitable for use in the present invention arepharmaceutically acceptable penetration enhancers. A pharmaceuticallyacceptable penetration enhancer can be applied to the skin of a humanpatient without detrimental effects (i.e., has low or acceptabletoxicity at the levels used).

Penetration enhancers suitable for use in the present invention include,but are not limited to, enhancers from any of the following classes:fatty alcohols, fatty acids (linear or branched); terpenes (e.g., mono,di and sequiterpenes; hydrocarbons, alcohols, ketones); fatty acidesters, organic acids, ethers, amides, amines, hydrocarbons, alcohols,phenols, polyols, surfactants (anionic, cationic, nonionic, bile salts).

Penetration enhancers can be characterized by a variety of physical, aswell as structural, properties. For example, in some embodiments of thepresent invention, a penetration enhancer component of the solventsystem has a molecular weight not greater than 400, is liquid at roomtemperature, and has a vapor pressure less than 10 mm Hg at 32° C.Examples, for illustration and not limitation, of such compounds areprovided in Table 1. (Tables 1-5 are provided at the end of thespecification.)

In some embodiments of the present invention, a penetration enhancercomponent of the solvent system has a molecular weight not greater than400, is liquid at room temperature, but which have a vapor pressuregreater than 10 mm Hg. Penetration enhancers of this type usuallyconstitute less than 100% (v/v) of the solvent system, more usually notmore than 95% of the solvent system, even more usually not more than75%, still more usually not more than 50% of the solvent system, andmost usually these penetration enhancers contribute no more than about30% (v/v) of the solvent system. Examples, for illustration and notlimitation, of such compounds are provided in Table 2.

In some embodiments of the present invention, a penetration enhancercomponent of the solvent system is not liquid at room temperature (e.g.,myristyl alcohol). Such “solid penetration enhancers” are not generallyused as the sole component of the solvent system. However, a solventsystem that contains a mixture of components can include a solidpenetration enhancer(s), so long as the solid penetration enhanceritself is in a solution. For example, a solvent system containing 95%diethylene glycol monoethyl ether and 5% myristyl alcohol (where themyristyl alcohol is in solution) can be used. Penetration enhancers ofthis type usually constitute less than 100% (v/v) of the solvent system,more usually not more than 95% of the solvent system, even more usuallynot more than 75%, still more usually not more than 50% of the solventsystem, and most usually these penetration enhancers contribute no morethan about 30% (v/v) of the solvent system. Examples, for illustrationand not limitation, of such compounds are provided in Table 3.

In some embodiments of the present invention, a penetration enhancercomponent of the solvent system has a molecular weight less than 50.Penetration enhancers of this type usually constitute less than 100%(v/v) of the solvent system, more usually not more than 95% of thesolvent system, even more usually not more than 75%, still more usuallynot more than 50% of the solvent system, and most usually thesepenetration enhancers contribute no more than about 30% (v/v) of thesolvent system.

In some embodiments of the present invention, a penetration enhancercomponent of the solvent system is a surfactant. In certain embodiments,the proportion of the solvent system that made up of surfactants is notmore than 5% (v/v).

In some embodiments of the present invention, a penetration enhancercomponent of the solvent system is a urea. In certain embodiments, theproportion of the solvent system that made up of ureas is not more than10% (v/v), or alternatively not more than 5% (v/v).

In one embodiment, the solvent system contains only one penetrationenhancer. In a related embodiment, the solvent system contains twopenetration enhancers, three penetration enhancers, four penetrationenhancers, five penetration enhancers, or more than five penetrationenhancers. Usually the solvent system contains one to four penetrationenhancers.

Penetration enhancers particularly suited for use in the presentinvention include fatty alcohols and terpenes.

Examples of useful fatty alcohols useful as penetration enhancersinclude oleyl alcohol, elaidyl alcohol, linoleyl alcohol, elaidolinoleylalcohol, linolenyl alcohol, elaidolinolenyl alcohol, cetyl-stearylalcohol, lauryl-myristyl alcohol, octyl-decyl alcohol, octyl alcohol,decyl alcohol, myristyl alcohol, cetyl alcohol, stearyl alcohol, laurylalcohol, 2-lauryl alcohol, ricinol alcohol, tallow alcohol, and caprylicalcohol.

Terpenes have molecular formulas (C_(n)H_(2n-4)), and are classifiedaccording to the number of isoprene units. Terpenes can occurtheoretically in the following four configurations: (1) Three doublebonds and no cycle (e.g., ocimene and myrecene), (2) Two double bondsand one cycle (e.g., limonene and Carveol), One double bond and twocycle (e.g., α-pinene or β-pinene and Limonene oxide). Sesquiterpeneshave formula (C_(n)H_(2n-6)) and can theoretically occur in variety ofconfigurations. Given the diverse nature of Terpenes and lack of strictdefinition of classification of terpenes, the foregoing description ofterpenes and sesquiterpenes is not intended to restrict the invention inany manner.

Other examples include monoterpenes (2 isoprene units), sesquiterpenes(3 isoprene units), diterpenes (4, isoprene units), triterpenes (6isoprene units) and tetraterpenes (8 isoprene units). Examples ofmonoterpenes are: nerol, citral, camphor, menthol. Examples ofsesquiterpenes are: nerolidol, farnesol. Examples of diterpenes are:phytol, vitamin A1. Squalene is an example of a triterpene, and carotene(provitamin A1) is a tetraterpene. Examples, for illustration and notlimitation, of terpenes useful as penetration enhancers includemethylnoneonoic acid and methylnoneonoyl alcohol, oxide, cyclopenteneoxide D-limonene, β-carene, α-terpineol, terpinen-4-ol, carvone,pulegone, piperitone, menthone, and 1,8-cineole. In one embodiment,terpenes used in the practice of the invention have a molecular weightless than 600. In one embodiment, terpenes used in the practice of theinvention have a molecular weight greater than 100. In one aspect, thepresent invention provides a method of increasing the amount of atopically applied TRPV1 agonist that enters the epidermal and dermallayers by topically applying the molecule in a composition comprising aterpene. In an embodiment, the invention provides a pharmaceuticalcomposition comprising a terpene and a TRPV1 agonist. in an embodiment,the TRPV1 agonist is capsaicin. In an embodiment, the terpene ismethylnonenoic acid or methylnonenoyl alcohol. In another embodiment,the terpene is selected from the group consisting of α-pinene oxide,limonene oxide, cyclopentene oxide _(D)-limonene, α-pinene, β-carene,α-terpineol, terpinen-4-ol, carvol, carvone, pulegone, piperitone,menthone, and 1,8-cineole.

One useful penetration enhancer of the solvent system is menthone. Insome versions of the invention, the solvent system contains at leastabout 50%, at least about 70%, at least about 80%, at least about 90%,at least about 95%, or about 100% menthone.

Another useful penetration enhancer of the solvent system ismethylnonenoic acid. In some versions of the invention, the solventsystem contains at least about 50% (v/v), at least about 70%, at leastabout 80%, or at least about 90% methylnonenoic acid. In one aspect, theinvention provides a pharmaceutical composition containing a TRPV1agonist (e.g., capsaicin) and methylnonenoic acid.

Another useful penetration enhancer of the solvent system ismethylnonenoyl alcohol. In some versions of the invention, the solventsystem contains at least about 50% (v/v), at least about 70%, at leastabout 80%, or at least about 90% methylnonenoyl alcohol. In one aspect,the invention provides a pharmaceutical composition containing a TRPV1agonist (e.g., capsaicin) and methylnonenoyl alcohol. In another aspect,the invention provides a method for increasing delivery of a TRPV1agonist to a tissue (e.g., epidermis and/or dermis) by administering acomposition containing the agonist and methylnonenoyl alcohol.

The use of methylnonenoyl alcohol and methylnonenoic acid to enhancedermal penetration of therapeutically active agents such as capsaicinhas not previously been described. In one aspect, the present inventionprovides a method of increasing the amount of a topically appliedmolecule that enters the epidermal and dermal layers by topicallyapplying the molecule in a composition comprising methylnonenoyl alcoholor methylnonenoic acid. In an embodiment, the molecule is atherapeutically active agent. In an embodiment, the molecule is a TRPV1agonist.

Another useful penetration enhancer of the solvent system is cetylalcohol. In some versions of the invention, the solvent system containsat least about 10% (v/v), at least about 20%, at least about 30%, or atleast about 40% cetyl alcohol.

Another useful penetration enhancer of the solvent system is oleylalcohol. In some versions of the invention, the solvent system containsat least about 50% (v/v), at least about 70%, at least about 80%, or atleast about 90% oleyl alcohol.

Another useful penetration enhancer of the solvent system is propyleneglycol. In some versions of the invention, the solvent system containsat least about 50% (v/v), at least about 70%, at least about 80%, or atleast about 90% propylene glycol.

Another useful penetration enhancer of the solvent system is diethyleneglycol monoethyl ether (DGME), which is commercially available asTranscutol® (Gattefossé Corp., Paramus, N.J.). In some versions of theinvention, the solvent system contains at least about 70% (v/v), atleast about 80%, at least about 90%, at least about 95%, or at leastabout 99% diethylene glycol monoethyl ether. In some embodiments of theinvention, the solvent system does not contain DGME or DGME constitutesnot more than 95% of the solvent system, alternatively not more than 75%of the solvent system, alternatively not more than 50% of the solventsystem, and alternatively not more than about 30% (v/v) of the solventsystem.

In some embodiments, the solvent system contains one, or two or more ofthe following penetration enhancers: menthone, methylnonenoic alcohol,methylnonenoic acid, oleyl alcohol, isopropyl myristate, dimethylisosorbide, and propylene glycol.

Exemplary solvent systems contain the following combinations ofpenetration enhancers, with zero, or optionally one, two, three or morethan three additional penetration enhancers: d-pipertone and oleic acid;l-menthone and oleic acid; l-menthone and ethyl oleate; l-menthone andbenzyl alcohol; ethylene glycol and l-menthone; benzyl alcohol and oleylalcoholic; l-menthone and cetyl alcohol; 1,3-butanediol and oleic acid;diethylene glycol monoethyl ether and l-menthone; ethelyne glycol andoleic acid; isopropyl myristate; oleyl alcohol and 1-3, butandiol;l-menthone and isopropyl butyrate; l-menthone and 1,3-butanediol;n-hexane and oleic acid; menthone and methanol; methylnonenoic acid andn-hexane; oleyl alcohol and propylene glycol; methylnonenoic alcohol anddimethylacetamide and Brij.

Exemplary solvent systems are (i) menthone 90% (v/v) plus methanol 10%(v/v); (ii) methylnonenoic acid 95% plus n-hexane 5%; (iii) oleylalcohol 20% plus propylene glycol 80%; (iv) methylnonenoic alcohol 94%plus dimethylacetamide 5% plus Brij-35 1%. Capsaicin is expected to bestable for extended periods in these formulations, which are highlylipophilic and absorb little water. Additional exemplary solventsystems, for illustration and not limitation, are shown in Table 6.

When the solvent system contains more than one penetration enhancer, itis sometimes the case that one of the penetration enhancers predominatesin the mixture. For example, in embodiments of the invention, the ratioof the predominant penetration enhancer to the sum of the otherpenetration enhancers in the solvent system is at least about 2:1, atleast about 3:1; at least about 5:1, at least about 8:1, at least about9:1 (v/v) or at least about 20:1. In one embodiment, the predominantpenetration enhancer is diethylene glycol monoethyl ether. In oneembodiment, the predominant penetration enhancer is menthone.

Exemplary penetration enhancers include stearyl alcohol, oleyl alcohol,linoleyl alcohol, linolenyl alcohol, caprylic alcohol, decyl alcohol,lauryl alcohol, Propylene glycol, polyethylene glycol, ethylene glycol,diethylene glycol, triethylene glycol, ethoxy digkycol, dipropyleneglycol, glycerol, propanediol, butanediol, pentanediol, hexanetriol2-lauryl alcohol, myristyl alcohol, cetyl alcohol, capric acid, lauricacid, myristic acid, stearic acid, oleic acid, caprylic acid, valericacid, heptanoic acid, pelagonic acid, caproic acid, isovaleric acid,neopentanoic acid, trimethyl hexanoic acid, neodecanoic acid, isostearicacid, neoheptanoic acid, neononanoic acid, isopropyl n-decanoate,isopropyl palmitate, octyldodecyl myristate, ethyl acetate, butylacetate, methyl acetate, isopropyl n-butyrate, ethylvalerate,methylpropionate, diethyl sebacate, ethyl oleate, isopropyl n-hexanoate,isopropyl myristate, urea, dimethylacetamide, diethyltoluamide,dimethylformamide, dimethyloctamide, dimethyldecamide,1-hexyl-4-methoxycarbonyl-2-pyrrolidone,1-lauryl-4-carboxy-2-pyrrolidone, 1-methyl-4-carboxy-2-pyrrolidone,1-alkyl-4-imidazolin-2-one, 1-methyl-2-pyrrolidone, 2-pyrrolidone,1-lauryl-2-pyrrolidone, 1-hexyl-4-carboxy-2-pyrrolidone,1-methyl-4-methoxycarbonyl-2-pyrrolidone,1-lauryl-4-methoxycarbonyl-2-pyrrolidone, dimethylsulfoxide,decylmethylsulfoxide, N-cocoalkypyrrolidone,N-dimethylaminopropylpyrrolidone, N-tallowalkylpyrrolidone,N-cyclohexylpyrrolidone, 1-farnesylazacycloheptan-2-one,1-geranylgeranylazacycloheptan-2-one, fatty acid esters of-(2-hydroxyethyl)-2-pyrrolidone, 1-geranylazacycloheptan-2-one,1-dodecylazacycloheptane-2-one (Atone®),1-(3,7-dimethyloctyl)azacycloheptan-2-one,1-geranylazacyclohexane-2-one,1-(3,7,11-trimethyldodecyl)azacyclohaptan-2-one,1-geranylazacyclopentan-2,5-dione, 1-farnesylazacyclopentan-2-one,benzyl alcohol, butanol, pentanol, hexanol, octanol, nonanol, decanol,ethanol, 2-butanol, 2-pentanol, propanol, diethanolamine,triethanolamine; hexamethylenelauramide and its derivatives,benzalkonium chloride, sodium laurate, sodium lauryl sulfate;cetylpyridinium chloride, citric acid, succinic acid, salicylic acid.sylicylate Cetyltrimethyl ammonium bromide, tetradecyltrimethylammoniumbromide; octadecyltrimethylammonium chloride; dodecyltrimethylammoniumchloride, hexadecyltrimethylammonium chloride, Span 20, Span 40, Span60, Span 80, Span 85, Poloxamer231, Poloxamer182, Poloxamer184), Brij30, Brij 35, Brij 93, Brij 96, Span 99, Myrj45, Myrj51, Myrj52, Miglyol840, glycholic, sodium salts of taurocholic, lecithin, sodium cholate,desoxycholic acids, D-limonene, α-pinene, β-carene, α-terpineol,terpinen-4-ol, carvol, carvone, pulegone, piperitone, Ylang ylang,menthone, anise, chenopodium, eucalyptus, limonene oxide, α-pineneoxide, cyclopentene oxide, 1,8-cineole, cyclohexene oxide, N-heptane,N-octane, N-nonane, N-decane, N-undecane, N-dodecane, N-tridecane,N-tetradecane, N-hexadecane, and essential oils (e.g., tea tree oils).

As discussed below, in some embodiments the solvent system may containelements other than the penetration enhancer(s), such as water or otherexcipient. In some embodiments, the penetration enhancer (if the solventsystem contains only one penetration enhancer) or penetration enhancerstogether (if the solvent system contains more than one penetrationenhancer) account for at least about 20% of the volume of the solventsystem. Often the penetration enhancer(s) account for at least about 40%of the volume of the solvent system, often at least about 50% of thevolume of the solvent system, often at least about 75% of the volume ofthe solvent system, often at least about 80% of the volume of thesolvent system, often at least about 90% of the volume of the solventsystem, often at least about 95% of the volume of the solvent system,often at least about 98% of the volume of the solvent system, sometimesat least about 99% of the volume of the solvent system, sometimes atleast about 99.5% of the volume of the solvent system, and sometimes100% of the volume of the solvent system.

5.1.2 Other Components of the Solvent System

In some embodiments of the invention, the solvent system contains liquidcomponents (water, saline, etc.) in addition to a penetration enhanceror combination of penetration enhancers. In some embodiments of theinvention, the solvent system is biphasic and the TRPV1 agonist issoluble in at least one phase. In an embodiment solvent system ismonophasic.

5.2 TRPV1 Agonist and/or Other Therapeutically Active Agents

5.2.1 Administration of TRPV1 Agonists

Exemplary TRPV1 agonists are described above (Section 3). In someembodiments of the invention the administered composition also containsone or more additional therapeutically active agents that areco-administered with the TRPV1 agonist(s).

Using the methods and compositions disclosed herein, therapeuticallyeffective amounts of TRPV1 agonists such as capsaicin can beadministered (e.g., topically) to a subject much more rapidly than ispossible using conventional formulations. Capsaicin-mediated therapeuticbenefits (including reduction of the density of cutaneous or mucosalnociceptors and amelioration of capsaicin-responsive conditions and/ortheir characteristic symptoms) can be achieved by administration ofcapsaicin at a lower concentration and/or for a shorter period thanheretofore believed or demonstrated. For some applications it will bedesirable to use a relatively high concentration for a short time, whilein other cases there will be advantages to using a lower concentration.The concentration of TRPV1 agonist in the composition can range from0.05 to 60% w/v, depending on the specific TRPV1 agonist, solvent systemused, and desired outcome.

In some embodiments, the concentration of TRPV1 agonist in thecomposition of the invention is in the range about 1% (w/v) to about40%, about 5% to about 25%, about 10% to about 20%, or about 15%.

In one embodiment, the concentration of TRPV1 agonist is less than about3% (w/v). In some embodiments, the concentration of TRPV1 agonist in thecomposition of the invention is in the range about 0.001% to about 20%,about 0.05% to about 20%, about 0.1% to about 10%, or about 0.1% toabout 5%. In one embodiment, the concentration of TRPV1 agonist is lessthan about 3%. Other exemplary ranges are from about 0.001% to about0.09%, about 0.001% to about 0.05%, about 0.001% to about 0.5%, fromabout 0.01% to about 1%, about 1% to about 5%, about 1% to about 10%,about 2% to about 7%, and about 2% to about 5%. In various embodiments,the TRPV1 agonist is present at a concentration in a range bounded by alower limit of 0.001%, 0.010%, 0.05%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%,5%, 6%, 7%, 7.5%, or 10% and an independently selected upper limit of0.010%, 0.05%, 0.5%, 1% 2%, 3%, 4%, 5%, 6%, 7%, 7.5%, 10%, 20%, 30%,40%, 50% or 60% (where the upper limit is greater than the lower limit).

In one embodiment, capsaicin (or a capsaicin analog) at a concentrationof less than 5% (w/v), less than about 3%, less than about 2%, less thanabout 1%, or less than about 0.5%.

Usually, the concentration of TRPV1 agonist is such that atherapeutically effective dose of the TRPV1 agonist can be delivered ina volume that is conveniently applied to the skin of the subject (e.g.,usually a volume of from about 5 μL to 50 μL per 1 cm², often a volumeof about 50 μL per 1 cm², often about 25 μL per 1 cm², often about 10 μLper 1 cm², often between about 5 μL and 25 μL per 1 cm² or about 5 μLand 10 μL per 1 cm²).

In one embodiment, a composition of the invention contains more than oneTRPV1 agonist (e.g., two, three, four, or more TRPV1 agonists). In oneembodiment, the composition contains capsaicin and another TRPV1agonist. Usually the combined concentration of TRPV1 agonists in thecomposition is 0.05 to 60% w/v, more often 0.05% to 10%, frequently 0.1%to 15%, 0.1% to 10%, or 1% to 10%. In one embodiment, the composition ofthe invention contains a single TRPV1 agonist. In one embodiment, theTRPV1 agonist is capsaicin.

5.3 Therapeutically Active Agents Other than TRPV1 Agonists

In some embodiments, the administered composition includes one or moreadditional therapeutically active agents (“TAA”) that areco-administered with the TRPV1 agonist(s). As used herein, the termtherapeutically active agent refers to an agent, other than a TRPV1agonist, with a biologically desirable activity that can be administeredto a subject by topical application to the skin, eyes, or to oral ornasal mucosa. Typically, the TAA has a molecular weight less than 1000,often less than 500. It will be understood that penetration enhancers,vehicles, solvents and the like are not examples of TAAs.

In one embodiment an additional therapeutically active agentco-administered with the TRPV1 agonist(s) is a local anesthetic.Exemplary local anesthetics include, without limitation,acetamidoeugenol, alfadolone acetate, alfaxalone, amucaine, amolanone,amylocaine, benoxinate, benzocaine, betoxycaine, biphenamine,bupivacaine, burethamine, butacaine, butaben, butanilicaine, buthalital,butoxycaine, carticaine, 2-chloroprocaine, cocaethylene, cocaine,cyclomethycaine, dibucaine, dimethisoquin, dimethocaine, diperadon,dyclonine, ecgonidine, ecgonine, ethyl aminobenzoate, ethyl chloride,etidocaine, etoxadrol, β-eucaine, euprocin, fenalcomine, fomocaine,hexobarbital, hexylcaine, hydroxydione, hydroxyprocaine,hydroxytetracaine, isobutyl p-aminobenzoate, ketamine, leucinocainemesylate, levoxadrol, lidocaine, mepivacaine, meprylcaine,metabutoxycaine, methohexital, methyl chloride, midazolam, myrtecaine,naepaine, octacaine, orthocaine, oxethazaine, parethoxycaine,phenacaine, phencyclidine, phenol, piperocaine, piridocaine,polidocanol, pramoxine, prilocaine, procaine, propanidid, propanocaine,proparacaine, propipocaine, propofol, propoxycaine, pseudococaine,pyrrocaine, risocaine, salicyl alcohol, tetracaine, thialbarbital,thimylal, thiobutabarbital, thiopental, tolycaine, trimecaine, andzolamine, and combinations thereof.

In other embodiments, the additional therapeutically active agent(s)co-administered with the TRPV1 agonist(s) are other than a localanesthetic. For example and not limitation, the TAA can be a steroid, anon-steroidal anti-inflammatory drug (e.g., ibuprofen, ketoprofen,flurbiprofen, naproxen, ketorolac and diclofenac), opioid analgesic(e.g., fentanyl and buprenorphine), antineoplastic agent (e.g.,5-flurouracil), or any of a variety of other drugs. Generally, the TAAis an agent for which local (e.g., dermal) administration is desired.

The concentration of the TAA in the composition can range from 0.05 to60% w/v, depending on the specific TAA and the solvent system used. Theconcentration of TAA in the composition is usually in the range about0.05% to about 10%, often in the range about 0.1% to about 10%, and mostoften in the range about 0.1% to about 5%. Usually, the concentration issuch that a therapeutically effective dose of the TAA can be deliveredin a volume that is conveniently applied to the skin of the subject(e.g., usually a volume of between about 0.05 mL and 10 mL, more oftenbetween about 0.1 mL and 5 mL, even more often between about 0.25 mL and1 mL.)

In various embodiments, the TRPV1 agonist is at a concentration in arange bounded by a lower limit of 0.001% (w/v), 0.010%, 0.05%, 0.1%,0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 7.5%, or 10% and an independentlyselected upper limit of 0.001%, 0.010%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%,4%, 5%, 6%, 7%, 7.5%, 10%, 20%, 30%, 40%, 50% or 60% (where the upperlimit is greater than the lower limit) and the local anesthetic is at aconcentration in a range bounded by a lower limit of 0.1%, 0.5%, 1%, or2% and an independently selected upper limit of 0.5%, 1%, 2%, 5% or 10%(where the upper limit is greater than the lower limit). In anembodiment, the local anesthetic is tetracaine. Usually, the combinedconcentration of TRPV1 agonist and other TAA(s) is in the range 0.05 to60% w/v, more often 0.05% to 10%, and frequently 0.1% to 10%.

5.4 Administration of Therapeutically Active Agents Other than TRPV1Agonists

In a related aspect of the invention, a therapeutically active agentother than a TRPV1 agonist is administered as a composition thatincludes a solvent system as described elsewhere herein with respect toadministered compositions of the invention. Thus, the TAA compositionhas the following:

-   -   1. A solvent system in which the TAA is soluble, containing at        least one penetration enhancer;    -   2. One or more TAAs;    -   3. Additional components that, if present, account for not more        than 5% (w/v) of the composition.        In preferred embodiments, the solvent system is characterized by        having a high concentration of penetration enhancer(s), such as        at least about 50%, at least about 75%, at least about 85%, at        least about 90%, at least about 95%, at least about 99%, at        least about 99.5%, or 100%.

Exemplary TAAs that can be administered using the compositions includethose listed in Section 5.2, supra, and are typically agents for whichlocal (e.g., dermal) administration is desired and are often drugs thatact at a site very close to the site of administration (e.g., localanesthetics).

5.5 Other Components of the Administered Composition

The composition may also contain stabilizers, pH modifiers, colorantsand fragrance or other compounds. These components account for less thanabout 5% (w/v) of the composition, more often less than about 2%, andoften less than about 1% or even about 0.5% of the composition.

Stabilizers useful in the compositions include materials that aid inensuring a stable composition (e.g., maintenance of viscosity over time,maintenance of pH over time, or maintenance of purity, appearance,homogeneity, and/or color over time) and/or prevent growth of bacteriaor other microorganisms and/or to maintain the chemical stability of theagonist or other therapeutically active agent against hydrolysis,oxidation, thermal or photolytic degradation. Exemplary stabilizersinclude antioxidants, chelators, preservatives (e.g., disodium edetate,beta-carotene, tocopherols, beta-tocopherols, tocopherol acetate, octylgallate, ascorbyl palmitate, butylated hydroxyanisole, butylatedhydroxytoluene); antimicrobial agents (i.e., include any compoundeffective in reducing or preventing build up of microbial load in theformulation, e.g., parabens, methylparaben, propylparaben, butylparaben,methyl salicylate, phenethyl alcohol, and resorcinol); and other agents(see, e.g., U.S. Pat. Nos. 6,013,270 and 6,390,291).

5.6 Optional Agents not Generally Required and Sometimes Omitted orPresent in Only Small Amounts

Topically administered agents are often administered in moderate to highviscosity forms (e.g., as a gel, lotion or cream) or via a topical ortransdermal patch. In some embodiments of the invention, a compositionof the invention is an “immediate-release composition” in which all ormost (i.e., entire dose) of the therapeutic agent is available at thesite of administration (e.g., skin, mucosa or epithelial surface) ratherthan administered over a sustained period. In some embodiments, thecomposition is a low viscosity composition (i.e., TRPV1 agonists aredelivered in a low viscosity composition). As used in this context, alow viscosity composition is one having a viscosity less than about 5000centipoise (cps), sometimes less than about 1000 cps, less than about500 cps, less than about 100 cps, less than about 50 cps, less thanabout 40 cps, less than about 20 cps, or less than about 10 cps whenmeasured before application to the skin, or alternatively, when measuredat 32° C. (skin temperature). Viscosity can be measured using standardmethods, e.g., by cone-and-plate viscometer or coaxial-cylinderviscometer. Transdermal patches (e.g., reservoir, matrix and microreservoir patches) are widely used for drug delivery, and are generallyocclusive and/or adherent devices. In addition, compositions deliveredby patches are by nature “delayed release” compositions. Administrationof a therapeutic agent without the use of an occlusive patch device isreferred to as “non-occlusive” administration or contacting.Administration of a therapeutic agent without the use of a skin-adherentdevice is referred to as “non-adherent” administration or contacting. Atherapeutic agent administered without the use of a delayed releasemechanism is referred to as an “immediate release” composition. Incertain embodiments of the present invention, TRPV1 agonists aredelivered without the use of a transdermal patch device and/or withoutthe use of any occlusive and/or without the use of adhesive and/or asimmediate delivery compositions.” In an embodiment, the TRPV1 agonist ofthe administered composition enters tissue passively (i.e., without theuse of an occlusive material to increase speed of entry).

Thus, in some embodiments of the invention, the administered compositionis free from agents generally added to compositions for topicaladministration to increase viscosity or otherwise maintain contact of atopically administered agent to the skin surface for an extended periodand/or added to modify flow characteristics to facilitate application toa defined area, or, if present, such agents are present at only very lowamounts (e.g., less than about 3% (w/v), less than about 1%, usuallyless than about 0.5%, and most usually less than 0.1%). For example, insome embodiments, ethyl cellulose can be included, if at all, at aconcentration of less than 1%, more usually less than 0.5%, most usuallyless than 0.1% or less than 0.05%.

Thus, in some embodiments, the administered composition has very lowviscosity and is free, or substantially free (which, in this context,means less then 0.1% w/v) of thickeners and gelling agents such asalkene copolymers (e.g., butylene-ethylene-styrene copolymer orethylene-propylene-styrene copolymer), cross-linked polyacrylatepolymers, carboxylic acid polymers, polyacrylamide polymers, acrylicacid/ethyl acrylate copolymers, carboxyvinyl polymers, Carbopol™ resins(colloidally water-soluble polyalkenyl polyether crosslinked polymer ofacrylic acid cross-linked with a cross-linking agent such as polyallylsucrose or polyallyl pentaerythritol), acacia, agar, alginic acid,aluminum monostearate, attapulgite (activated or colloidal activated),bentonite, purified bentonite, bentonite magma, carboxymethylcellulosecalcium, carboxymethylcellulose sodium, carboxymethylcellulose sodium12, carrageenan, microcrystalline cellulose, carboxymethylcellulosesodium, dextrin, gelatin, guar gum, hyaluronic acid, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,magnesium aluminum silicate, methylcellulose, pectin, polyethyleneoxide, polyvinyl alcohol, povidone, propylene glycol alginate, silicondioxide, colloidal silicon dioxide, viscous silicone oil (>5000 cps),silicone-based gels, sodium alginate, tragacanth, xanthan gum, andaluminum silicates.

5.7 The Form of the Administered Composition

In some embodiments of the invention, the administered composition isform suitable for administration to a subject (e.g., human patient). Inone embodiment, the composition is provided in a unit dosage form ormultiunit dosage form. As used herein, a unit dosage form means anamount of the administered composition suitable for a singleadministration to a single subject in need of treatment, and multiunitdosage form means an amount of the administered composition suitable fora multiple administrations (e.g., usually from 2 to 10 administrations,more usually from 2 to 5 administrations, even more usually from 2 to 4administrations and most usually 2 or 3 administrations). The unitdosage form and multiunit dosage form may be in the form of a liquidsolution in one or more vials or similar containers. Typically thecontent of each vial will be between 0.1 mL and 100 mLs, more often from0.5 to 50 mL, more often from 1 to 10 mL. In some embodiments, the unitdosage or multiunit dosage is contained in a syringe, a dropper, apipette or other liquid delivery device. In some embodiments, the unitdosage or multiunit dosage is contained in a spray or aerosol deliverydevice. In some embodiments, the unit dosage or multiunit dosage iscontained in a syringe. In some embodiments the unit dosage or multiunitdosage is in the form of a towellete or other absorbent materialimpregnated with the administered composition. In one embodiment, eachunit dosage or multiunit dosage is individually packaged in a packagesuitable for storage and/or shipping.

6. Properties of Administered Composition and Solvent System

This section describes additional properties of certain administeredcompositions that may be used in the practice of the invention. Theseproperties may be used in selecting combinations of penetrationenhancer(s) and other composition components for optimized use with aparticular type and concentration of agonist. However, the usefulcompositions of the invention are not limited to compositions having allof the exemplary properties described below.

6.1 the Administered Composition and Solvent System May be LiquidSolutions in which the TRPV1 Agonist(s) are Soluble

As noted above, the TRPV1 agonist is dissolved in the solvent system inan amount and at a concentration that will vary according to theparticular agonist, other TAAs present, the purpose of the composition,and the desired dose. In a preferred embodiment, will be appreciated,however, that in the present invention, the TRPV1 agonist is fully insolution in the solvent system (e.g., present in an amount that is lowerthan the saturation limit of the TRPV1 agonist in the solvent system).Shaking, heating, sonication and the like may be used to drive anagonist into solution, so long as the agonist remains in solution atroom or skin temperature after preparation.

The compositions of the present invention may be homogenous solutionswith substantially all of the TRPV1 agonist dissolved in the composition(and often dissolved in the solvent system component of thecomposition), and substantially no suspended or undissolved particles ofagonist. While the present invention is not limited to compositionsdisplaying only particular maximum level of turbidity, those of skill inthe art will understand that generally, turbidity is minimal.

6.2 the Composition May be Applied as a Thin Film

Some compositions of the invention can be applied as a thin homogeneousfilm, which does not require occlusion, bioadhesives, or other additivesor devices to effect pharmacological action. The formulation may beapplied through physical mechanical means including swab, applicatorpad, syringe spreader, or like devices intended to apply liquids in athin film. Since the administered compositions of the invention aretypically applied to skin at a dose of about 10 μL per cm², sometimesranging up to 20 or 30 μL per cm², these applications result in acomposition film thickness of about 100-300 μm.

Generally, the compositions of the invention are applied in a liquidvolume of at least about 5 μl/cm² application area (e.g., skin ormucosal surface), often at least about 7.5 μl/cm² application area, atleast about 10 μl/cm² application.

The compositions result in a thin layer of a low viscosity homogeneousliquid adsorbed to micro skin-surface irregularities and flowing withbody shape. The coverage, wetting and intrinsic contouring given byliquid topical applications, allows for maximum surface exposure due torheological and thermodynamic properties of a low viscosity fluid. Thecompositions demonstrate expected behaviors such as an initial wettingsheen followed by gradual dissipation. Application as a thin film maycontribute to the ability of the formulations to depot in skin in veryshort application durations.

6.3 the Composition May Disappear Rapidly Following Application

Some compositions of the invention are characterized by rapid andsubstantially complete disappearance from the surface of the skinfollowing application. In one embodiment, for example, the compositionsubstantially disappears (e.g., is absorbed and/or is evaporated) withinabout 30 minutes (and more usually within 15 minutes, 10 minutes, oftenwithin about 5 minutes, often within about 2 minutes, and sometimes evenwithin 1 minute) after application of about 5 μL or 10 μL to skin (e.g.,forearm) per cm² skin area (e.g., 250 μL per 25 cm²). By “substantiallydisappears” is meant that the majority (usually at least about 75%, atleast about 90% or at least about 95%) of the composition appliedtopically has dispersed by absorption through the stratum corneum intothe epidermis or dermis of the skin and/or by evaporative processes(e.g., as assessed by disappearance from the surface site ofapplication, e.g., the skin surface is dry to the touch or as assessedby other quantitative or qualitative methods). In one embodiment thedisappearance is primarily or completely due to absorption (e.g., themajority, or even at least about 75%, of the composition appliedtopically has dispersed by absorption). In another embodiment, thedisappearance is primarily or completely due to absorption. In anembodiment, at least about 5 μL of the composition is delivered to(absorbed into) each cm² of the skin or other treated region withinabout 15 minutes.

6.4 the Penetration Rate of the Composition May be Greater than theEvaporation Rate

Some compositions of the invention are characterized by having apenetration rate that is greater than its evaporation rate. As usedherein, the term “penetration rate” refers to the rate at which thecomposition penetrates the barrier of the stratum corneum and isabsorbed into the skin. As used herein, the term “evaporation rate”refers to the rate at which the components of the formulation undergo aphase change from liquid to gaseous form. When the compositionevaporation rate is greater than its penetration rate, it is especiallylikely that significant agonist will remain on the skin surface. Stateddifferently, when the vapor pressure of the composition is high, andhence its evaporation rate exceeds percutaneous penetration, asignificant residue of therapeutic agent may remain as a residualdeposit on the skin surface.

The relative penetration and evaporation rates of a composition may bedetermined by a variety of methods including those described by B. W.Kemppainen and W. G. Reifeinrath in METHODS FOR SKIN ABSORPTION, CRCPublication 1990. Evaporation/permeation analysis systems are availablefrom Laboratory Glass Apparatus, Inc. Berkeley, Calif.

7. Methods of Making Compositions

The compositions of the invention can be made using conventionaltechniques. Materials can be combined in any order. Illustrativepreparation methods for certain forms of the composition are describedhereinbelow.

In one embodiment, the components are manufactured or formulated in fullcompliance with all Good Manufacturing Practice (GMP) regulations of theU.S. Food and Drug Administration using materials suitable foradministration to human subjects.

8. High Flux Rate Application

In another aspect, the invention provides a method of treating acapsaicin-responsive condition in a subject by topical administration ofa composition containing a TRPV1 agonist under conditions in which theagonist penetrates the stratum corneum at a high flux rate. Whenadministered at a high flux rate, the rapid exposure of nerve fibers tocapsaicin and/or rapid accumulation of capsaicin in the epidermis ordermis results in a substantial reduction in density of functionalcutaneous nociceptors.

Flux rate refers to the movement of a compound (e.g., TRPV1 agonist)across a barrier (e.g., stratum corneum) and has units ofweight/area/unit time (e.g., μg/cm²/10 minutes).

As used in this context, “a high flux rate” means a flux rate of atleast about 10 μg/cm²/15 minutes, preferably at least about 10 μg/cm²/10minutes, even more preferably 20 μg/cm²/15 minutes, or even at leastabout 35 μg/cm²/15 minutes or at least about 35 μg/cm²/10 minutes, andsometimes even at least about 50 μg/cm²/15 minutes, at least about 75μg/cm²/15 minutes, or at least about 100 μg/cm²/15 minutes. A “high fluxrate” also means a flux rate of at least about 1 μg/cm²/minute,preferably at least about 2 μg/cm²/minute, at least about 3.5μg/cm²/minutes, at least about 5 μg/cm²/minute, at least about 7.5μg/cm²/minute, or at least about 10 μg/cm²/minute.

In one embodiment, the invention provides a method of treating acapsaicin-responsive condition in a subject by topical administration ofa composition containing TRPV1 agonist at a concentration of less than5% (w/v), usually less than about 3%, often not more than about 1%,often less than about 1%, often less than about 0.5%, and sometimes lessthan about 0.1 or 0.05% under conditions in which the agonist penetratesthe stratum corneum at a high flux rate.

In one embodiment, the invention provides a method of treating acapsaicin-responsive condition in a subject by topically administering acomposition containing a TRPV1 agonist at a high flux rate, wherein saidadministering is for less than about 45 minutes, preferably less thanabout 30 minutes, often about less than about 15 minutes, sometimesabout 10 minutes or less, or even less than about 5 minutes. In anembodiment, the composition administered contains capsaicin at aconcentration of less than 5% (w/v), usually less than about 3%, oftennot more than about 1%, often less than about 1%, often less than about0.5%, and sometimes less than about 0.1 or 0.05%.

In one embodiment, the invention provides a method of substantiallyreducing the density of functional cutaneous nociceptors in a topicalarea of a subject by topically administering a composition containing aTRPV1 agonist at a high flux rate where administration is for less thanabout 45 minutes, preferably less than about 30 minutes, often aboutless than about 15 minutes, sometimes about 10 minutes or less, or evenless than about 5 minutes. In an embodiment, the compositionadministered contains TRPV1 agonist at a concentration of less than 5%(w/v), usually less than about 3%, often not more than about 1%, oftenless than about 1%, often less than about 0.5%, sometimes less thanabout 0.1, and sometimes less than about 0.05%.

9. Administration of Composition

9.1 Duration of Administration

Depending on the purpose of administration and/or the condition beingaddressed, administration of the compositions of the invention can havea variety of beneficial effects, including a reduction in nerve fiberfunctionality, a change in skin sensitivity, relief from pain, and otherbeneficial effects. Unexpectedly, these beneficial effects usually canbe accomplished by a relatively short exposure to the composition. Forexample, the duration of administration sufficient to result inbeneficial effect is usually less than one hour, more often less than 30minutes, sometimes less than about 15 minutes, and sometimes less thanabout 10 minutes, sometimes about 5 minutes or less, and sometimes lessthan about 2 minutes. Using the methods and compositions disclosedherein, therapeutically effective amounts of TRPV1 agonists such ascapsaicin can be topically administered to a subject much more rapidlyand is greater amounts than is possible using conventional formulations.

In one embodiment, the invention provides a method of reducing chronicpain in a subject by topical administration of a TRPV1agonist-containing composition of the invention for less than about 45minutes, usually less than about 30 minutes, often about less than about15 minutes, sometimes about 10 minutes or less, or even less than about5 minutes. Generally one or two administrations are sufficient toprovide persistent relief

As used herein, the duration of administration can refer to the timeelapsed between first application of the composition to the subject(e.g., by spraying onto skin, by immersion, or the like, and either (1)ending contact (e.g., removing an immersed body part from a bath,removing an applicator device from the skin, and the like; (2) cleaningthe region contacted with the composition to remove any residual agonist(e.g., using a cleaning solution as described below in Section 10); or(3) the point at which the composition has entirely disappeared from thesite of application (e.g., by absorption into the skin, evaporation, ora combination of both).

9.2 Administration to Skin

The compositions of the invention may be applied to the skin (or,alternatively, to mucous membrane) using a large variety of methods anddevices. For example and not illustration, compositions may beadministered using a sponge, aerosol, spray, brush, swab, or otherapplicator. In one embodiment, the applicator provides either a fixed orvariable metered dose application such as a metered dose aerosol, astored-energy metered dose pump or a manual metered dose pump. In anembodiment, the applicator device has measuring marks for assisting auser in determining the amount of the composition in the applicatordevice.

In one embodiment, the applicator is non-occlusive. In one embodiment,the applicator does not adhere to the skin and/or is not adhesive. Inone embodiment, the applicator is not a patch device. In one embodiment,the applicator is a patch device and does not contain a penetrationenhancer selected from the group butanediols, dipropylene glycol,tetrahydrofurfuryl alcohol, diethylene glycol dimethyl ether, diethyleneglycol monoethyl ether, diethylene glycol monobutyl ether, propyleneglycol, dipropylene glycol, carboxylic acid esters of tri- anddiethylene glycol, polyethoxylated fatty alcohols of 6-18 C atoms,2,2-dimethyl-4-hydroxymethyl-1,3-dioxolane, dipropylene glycol,1,3-butanediol, diethylene glycol monoethyl ether or2,2-dimethyl-4-hydroxymethyl-1,3-dioxolane.

In one aspect, the invention provides a system for treating acapsaicin-responsive condition and having (1) an administeredcomposition as described herein (2) an applicator device for applyingthe formulation to skin or a mucosal surface. In one embodiment, theapplicator device is pre-filled with the composition. In one embodiment,the administered composition is contained in a container separate fromthe device.

9.3 Instillation

In one embodiment, the administered composition is administered byinstillation. As used in this context, instillation means introducingthe composition into a part of the body other than the skin, by a methodother than injection. Examples of instillation are introduction into thebladder via catheter, introduction into the nasal cavity by spray,instillation into the urethra; and instillation into surgical wounds(e.g., to treat or prevent pain).

9.4 Administration by Injection

In some embodiments of the invention, formulations described herein areadministered by injection. For example, injection methods may be used todeliver TRPV1 agonist to specific nerve trunks, tissues, or other sitesin a subject. Advantageously, the administered compositions of theinvention deliver a large quantity of a TRPV1 agonist in a small dosevolume; small dose volumes entail reduced pain and tissue injury, andconvenience for health care providers. Further, in view of thesubstantial depot effect seen in skin for some administered compositionsof the invention, it is expected that the compositions, when injected,will depot in the area of injection, resulting in a low level ofsystemic exposure while providing a high local concentration of a TRPV1agonist. Injection of TRPV1 agonists into nerve trunks can be used toblock the incoming afferent pain signals from distal nociceptive nervefibers, thereby providing benefit for patients with neuropathic painsyndromes (see, Pertovaara, 1988, “Collateral sprouting of nociceptiveC-fibers after cut or capsaicin treatment of the sciatic nerve in adultrats” Neurosci Lett. 90:248-53). In other example, injection of TRPV1agonist into prostate tissue can be used to control prostate cancer byselectively killing cancers cells of prostatic origin (see, e.g., Morreet al., 1997, “NADH oxidase activity from sera altered by capsaicin iswidely distributed among cancer patients” Arch Biochem Biophys342:224-30; Szallasi et al., 2001, “Vanilloid receptor ligands: hopesand realities for the future” Drugs Aging. 18:561-73; Surh, 2002,“Anti-tumor promoting potential of selected spice ingredients withantioxidative and anti-inflammatory activities: a short review” FoodChem Toxicol. 40:1091-7; Van der Aa et al., 2003, “Interstitial cells inthe human prostate: a new therapeutic target? Prostate 56:250-5).

9.5 Administration as a Microemulsion

In one embodiment, the administered composition is applied or instilledin the form of a microemulsion. In one embodiment, the microemulsion isinstilled (introduced into) the bladder.

9.6 Other Administration Forms

It will be apparent to those of ordinary skill in the art that othermethods of administration are known. All suitable methods arecontemplated and encompassed by the invention. Other administrationforms include administration using fluid-filled microspheres (see, e.g.,U.S. Pat. No. 5,716,643; U.S. Pat. No. 6,264,988), liposomes, otherhollow vesicles, cyclodextrins, micellular, or bioerodible gels.

10. Removal of Residual TRPV1 Agonist

In one aspect, the invention provides method of contacting a surface(e.g., skin) with a TRPV1 agonist to reduce nerve fiber functionalityand/or treat capsaicin-responsive condition followed by the subsequentstep of removing any residual agonist from the surface. In anembodiment, the residual agonist is removed by rinsing. In a differentembodiment, the residual agonist removed by applying a cleaningcomposition in which the agonist is soluble and removing the cleaningcomposition. For illustration and not limitation, a suitable compoundmay be a polyethylene glycol (PEG)-based composition, such as aqueousgel containing 60 to about 99 percent w/w polyethylene glycol. In anembodiment, the cleaning agent is consists of PEG-300 (89.08%),polyacrylate thickening agent such as Carbopol 1382™ (1.0%), butylatedhydroxyanisole (0.02%), disodium edetate (0.1%), balance water; gel isat a pH of about 6.5. See, e.g., PCT publication WO04021990A2“Compositions and kits for the removal of irritating compounds frombodily surfaces.”

If desired, the amount of agonist remaining as a residue on a skinsurface can be determined using any of a variety of assays. For example,the residue can be removed from the skin by rinsing with a solvent inwhich the TRPV1 agonist is soluble, or wiping the application site witha swab, and the TRPV1 agonist weight, concentration or bioactivity inthe solvent or swab can be determined. Suitable procedures for thisdetermination will be apparent to those skilled in the art and withreference to the scientific literature. See, for example, Wang et al.,2001, Int J Pharm 14:89-104. In most assays, collection of samples ofthe residual amounts of drug on skin surface is conducted. One way ofsampling is by wiping the application site using a surgical grade gauzesponge lightly soaked with a solvent suitable for that particular drugsubstance. The wiping is performed such that each surface of sponge isexposed only once during single longitudinal stroke. The gauze sponge isthen placed on a sintered glass funnel and washed with an adequate knownamount of same solvent (the same known quantity is used to soak thegauze initially). The resulting wash is analyzed by a method(chromatography, UV spectroscopy or mass spectrum analysis) suitable forquantitative determination of drug substance in question. See, e.g., M.J. Shifflet and M. Shapiro Development of Analytical Methods toAccurately and Precisely Determine Residual Active PharmaceuticalIngredients and Cleaning Agents on Pharmaceutical Surfaces, AmericanPharmaceutical Review; Summer 2002. An alternative method of sampling isdescribed by Nanji A. et. al. 1987, J Toxicol. Clin Toxicol. 25:501-15(describing use of a suction probe to collect samples which weresubsequently loaded directly into a mass spectrometer by thermaldesorption).

11. Kits and Devices

In an aspect, the present invention provides a kit including (1) anadministered composition of the invention and materials for removingresidual agonist from the application surface (e.g., skin surface). Inan embodiment, the kit contains a PEG-based cleaning gel such as thosedescribed in PCT publication WO04021990A2.

Additionally, a kit of the invention may include an anesthetic,chemical-resistant disposal bags, applicators for applying the cleansingcomposition, towels or towelettes for removing the cleansing gel,gloves, eye protection, scissors, marking pens, and additional bodilysurface-cleansing agents such as alcohol swabs. In one embodiment, theanesthetic is lidocaine.

In an aspect, the invention provides a kit including (1) an administeredcomposition of the invention and (2) a material or device for deliveringthe composition. In an embodiment, the composition is contained in acontainer separate from the device. in one embodiment, the applicatordevice is a sponge, brush, or swab.

12. Exemplary Effects

Application of the TRPV1 agonists of the invention results in a varietyof beneficial physiological and/or therapeutic effects, some of whichexamples are described below.

12.1 Reducing the Density of Functional Nociceptive Nerve Fibers

As noted, in one aspect the invention provides a method of reducing thedensity of functional nociceptive nerve fibers (i.e., a reduction innerve fiber functionality, NFF) in a selected region of a subject bycontacting the region with a composition that contains a TRPV1 agonistand a solvent system containing one or more penetration enhancers, i.e.,an administered composition as described herein.

In some embodiments of the invention, contacting the area with theadministered composition for a specified period of time results in asubstantial reduction in density. A “substantial reduction” in densityor number of functional nociceptive nerve fibers means a reduction of atleast about 20%, at least about 25%, at least about 28%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 75%, and sometimes at least about 80% compared to anuntreated (control) region or subject. See Examples 2 and 3, infra. Insome embodiments, the specified period of time not more than about 10,15, 20, 30, or 45 minutes.

The density or number of functional nociceptive nerve fibers can bedetermined by a variety of methods. A particularly useful method isimmunostaining for protein gene product 9.5 (“PGP 9.5”) as described byNolano et al., 1999, J Neuroscience 81:135-45. Also see Kennedy et al.,1996, “Quantitation of epidermal nerves in diabetic neuropathy”Neurology 47:1042-48. A reduction in PGP 9.5 staining is indicative of areduction in NFF. Other methods for measuring functional or structuralinactivation of nerve fibers include substance P immunostaining,calcitonin-related-gene-product immunostaining, S100 immunostaining,immunostaining for neurofilament proteins and immunostaining for TRPV1receptors, e.g., using anti-TRPV1 receptor antibodies. Analysis ofimmunostaining is usually conducted between 2 and 7 days followingadministration of the TRPV1 agonist. In various embodiments nerve fiberdensity is measured 2, 3, 4, 5, 6 or 7 days after administration of thecomposition. In one embodiment, the density of functional nociceptivenerve fibers is measured 7 days after administration of the TRPV1agonist. Alternatively, methods other than immunostaining can be used toassess the density of cutaneous nociceptors.

Examples 2 and 3 illustrate assays for NFF following topical applicationof capsaicin or resiniferatoxin. Exposure to a TRPV1 agonist may lead toeither a frank reduction in the number of countable nerve fibers in theepidermis and dermis or to changes in the appearance of those nervefibers. When PGP 9.5 immunoassays are used to monitor NFF, only PGP9.5-positive nerve fibers with normal morphological appearance arecounted. Areas of skin affected by peripheral neuropathies arecharacterized by atypical swelling, varicosities or segmentation ofsmall diameter nerve fibers (nociceptors) (see, e.g., McArthur et al.,1998, “Epidermal nerve fiber density: normative reference range anddiagnostic efficiency” Arch Neurol. 55:1513-20; Herrmann et al., 2004,“Epidermal nerve fiber density, axonal swellings and QST as predictorsof HIV distal sensory neuropathy” Muscle Nerve. 29:420-7) and similarchanges in nociceptor morphology are believed to occur followingexposure of skin to topical capsaicin. In counting the number of nervefibers per mm of skin, only nerve fibers with normal morphologicalappearance are counted. Further, if a substantial number of nerve fibersin any skin section are swollen or have varicosities, such that it isnot possible to count fibers with normal appearance, and the section isassigned a value of 0 nerve fibers.

12.2 Sustained Reduction of Neuropathic Pain

In one aspect the invention provides a method, application of theadministered composition to a subject with neuropathic pain results in asustained or persistent diminution of symptoms (e.g., pain). In somecases, one or two administrations are sufficient to provide persistentrelief (i.e., relief for at least about four weeks, preferably at leastabout 8 weeks).

12.3 Reduction of Skin Sensitivity Following Administration of TRPV1Agonists (QST Assay)

Nociceptive nerve fibers normally respond to warm and cold thermalstimuli. Hence, changes in thermal thresholds are indicative of reducednociceptor function and can be used to measure NFF. A “substantialreduction” in NFF can be detected as a change in thermal threshold.Quantitative sensory testing (QST) can be used to detect changes inthermal thresholds, due either to diseases or exposure to TRPV1 agonists(Bjerring et al., 1989, “Use of a new argon laser technique to evaluatechanges in sensory and pain thresholds in human skin following topicalcapsaicin treatment” Skin Pharmacol. 2:162-67). Methods of QST are wellestablished in the scientific literature and known widely to thoseskilled in the art (Siao et al., 2003, “Quantitative sensory testing”Phys Med Rehabil Clin N Am. 14:261-86.) For example, a decreased abilityto detect the cold sensation produced by a metal roller (Therrelldevice, Somedic Production AB, Sollentuna, Sweden) pre-cooled to 12° C.can be measured. In various embodiments, the change in thermal thresholdis at least about 10%, at least about 20%, at least about 25%, at leastabout 28%, at least about 30%, at least about 40%, and sometimes atleast about 50% compared to an untreated (control) region or subject.

12.4 Administration of TRPV1 Agonists with Reduced Discomfort

It is generally known that administration of capsaicin produces anintense burning pain. Application of even relatively low concentrationsof capsaicin results in pain that is intolerable to many patients. Forthis reason, capsaicin may be administered following, or concurrentlywith, administration of anesthetic.

Surprisingly, it has been discovered that rapid administration of TRPV1agonists using the methods and compositions of the invention results inless pain or discomfort than administration of conventional capsaicinformulations containing much lower concentrations of capsaicin. Example4 shows that, surprisingly, 10% w/v capsaicin liquid formulationproduced statistically significant (p≤0.1) less pain response thanZostrix® cream when applied to rat vulva (Zostrix® cream is acommercially available 0.075% capsaicin formulation). Similarly, Example5 suggests that topical application of a 10% capsaicin liquidformulation in diethylene glycol monoethyl ether produced lessnocifensive behavior over a 90-minute observation period than didtopical application of a commercially available over-the-counterlow-concentration (0.1%) capsaicin cream.

It is contemplated that, using the methods of the present invention,moderate (>1%), and high (>3%) even a very high dose of capsaicin can beadministered to a patient without the requirement for pretreatment with,or coadministration of, anesthetic. Moreover, when anesthesia is usedbefore, during or after exposure to a TRPV1 agonist, less anesthetic orshorter exposure to anesthetic will be required to achieve the sameeffect on discomfort. Thus, in one aspect, the invention providesmethods for administration of a TRPV1 agonist such as capsaicin, at aconcentration of greater than 1% (w/v), greater than 2%, greater than3%, greater than 4%, greater than 5%, or greater than 6%, without theneed for anesthetic, using the compositions of the invention.

13. Therapeutic Uses of TRPV1 Agonist-Containing Compositions

This section describes use of the compositions of the invention.However, it will be understood that the examples in this section areprovided for illustration and not limitation. As noted above, capsaicinapplication has numerous therapeutic benefits, each of which can beeffectively treated using the methods of the invention. Conditions forwhich TRPV1 agonist treatment may be indicated include neuropathic pain(including pain associated with diabetic neuropathy, postherpeticneuralgia, HIV/AIDS, traumatic injury, complex regional pain syndrome,trigeminal neuralgia, erythromelalgia and phantom pain), pain producedby mixed nociceptive and/or neuropathic mixed etiologies (e.g., cancer),osteoarthritis, fibromyalgia, low back pain, inflammatory hyperalgesia,vulvar vestibulitis or vulvodynia, sinus polyps interstitial cystitis,neurogenic or overactive bladder, prostatic hyperplasia, rhinitis,surgery, trauma, rectal hypersensitivity, burning mouth syndrome, oralmucositis, herpes (or other viral infections), prostatic hypertrophy,dermatitis, pruritis, itch, tinnitus, psoriasis, warts, cancers(especially skin cancers), headaches, and wrinkles. Generally, the TRPV1agonist-containing compositions can be used to treat any condition forwhich topical administration of a TRPV1 agonist (e.g., capsaicin) isbeneficial.

13.1 Neuropathic Pain

Neuropathic pain, such as that associated with diabetic neuropathy orpostherpetic neuralgia, has proven particularly refractory to treatment.However, capsaicin has been demonstrated efficacious in treatment ofneuropathic pain. For example, inactivation of cutaneous nociceptors inthe epidermis and dermis induced by an 8% w/w capsaicin dermal patch hasdemonstrated clinical efficacy against postherpetic neuralgia, aprototypic neuropathic pain condition (see Backonja et al., “A SingleOne Hour Application of High-Concentration Capsaicin Patches Leads toFour Weeks of Pain Relief in Postherpetic Neuralgia Patients” AmericanAcademy of Neurology, 2003 (meeting abstract)). The compositions of thepresent invention are effective for treating such neuropathic pain.

The efficacy of specific compositions with respect to the ability torender cutaneous nociceptors persistently non-functional can bedetermined by immunohistochemical evaluation of the density of skinmarkers for cutaneous nociceptors, such as protein gene product 9.5 (PGP9.5). Standard methods can be utilized to quantify changes in PGP 9.5staining densities (see Nolano et al., 1999, J Neuroscience 81:135-45).Analysis can be made of punch biopsies taken 3 to 7 days followingtreatment with a formulation. Compositions of the present invention thatproduce loss of PGP 9.5 staining comparable or superior to the capsaicindermal patch (see Backonja et al., 2003) are expected to produce similaror superior analgesic activities.

A single administration of a TRPV1 agonist-containing (e.g.,capsaicin-containing) composition of the invention can be used toprovide significant and long lasting relief from chronic painconditions, particularly neuropathic pain and inflammatory pain. As usedin this context, significant pain relief means a reduction of at least15%, and sometimes at least 50%, relative to the pain that the patientinitially reported. Pain can be measured using routine techniques, suchas application of the Likert pain scale (see Guyatt et al., 1987, “Acomparison of Likert and visual analogue scales for measuring change infunction” J Chronic Dis. 40:1129-33). As used in this context, longlasting pain relief means relief for at least two weeks, usually atleast 1 month, and often relief for 3-6 months after administration. Itis expected that application of a composition comprising 1-10% (w/v)capsaicin for 2 to 60 minutes will provide significant relief. Usually,application for 30 minutes or less, such as 15 minutes or less, or even10 minutes or less will provide significant relief.

13.2 Inflammatory or Nociceptive Pain

The TRPV1 agonist are useful for amelioration of inflammatory ornociceptive pain, particularly that due to such conditions asosteoarthritis, rheumatoid arthritis, joint pain, surgery, trauma,bruises, abrasions, lower back pain, acute herpes zoster or cancers.

13.3 Cancers

TRPV1 agonist compositions of the invention are particularly valuablefor treatment of various types of cancers, e.g., skin cancers. Capsaicinhas been shown to prevent cancer cell growth and/or induce cancer cellapoptosis in vitro assays, using a variety of cancer cell lines (forreview, see Y-J Surh, 2002, “More Than Spice: Capsaicin in Hot ChiliPeppers Makes Tumor Cells Commit Suicide”, J Nat Cancer Inst,94:1263-65). According to the invention, the capsaicin or TRPV1 agonistcompositions of the invention are delivered directly to canceroustissues or cells, or to pre-cancerous cells (e.g., cells responsible forprostatic hyperplasia or uterine abnormalities). For treatment of skincancers, for example, the composition can be applied by topicalapplication or, alternatively, by injection or instillation. Becausecapsaicin enhances the percutaneous absorption of other compounds, thedelivery of a combination of capsaicin with anti-cancer compounds (e.g.,5-fluouracil) would be expected to be efficacious.

13.4 Oral Mucositis

TRPV1 agonist compositions of the invention are also used for treatmentof oral mucositis. Oral mucositis is a significant problem in patientsreceiving chemotherapy or radiation therapy. Estimates of oral mucositisin cancer therapy range from 40% receiving standard chemotherapy to 76%in bone marrow transplant patients. The efficacy of capsaicin fortreating oral mucositis has been described (Berger et al., 1995, J PainSymptom Manage 10:243-8). However, the pain relief provided by previousformulations was not complete for most patients and was of limitedduration. This may have been due to the nature of previous formulations,either their ability to induce pain or inability to precisely deliverhigh concentrations of capsaicin to the site of oral mucositis.

Administration of the compositions of the invention to lesions of oralmucosa would provide rapid and convenient delivery of capsaicin, orother TRPV1 agonists. Application of compositions could be achieved bymeans of a swab, spray, roller, syringe or other device.

13.5 Bladder Disorders

Instillation of capsaicin-containing solutions into the bladder has beenused to treat a variety of bladder disorders, including neurogenicbladder, interstitial cystitis, detrusor hyperreflexia and overactivebladder (for review, see Fowler et al., 2002, “Voiding and the SacralReflex Arc: Lessons from Capsaicin Instillation,” Scand J Urol NephrolSupple 210:46-50.). Although generally efficacious, these proceduresrequire at least 20 minutes of drug solution exposure, and producesignificant pain and discomfort for patients. See Chancellor et al.,1999, J Urol 162:3-11. The compositions of the present invention can beused to treat such bladder disorders. In one embodiment, the compositionis administered in the form of a microemulsion such as is describedinfra in Section 13 instilled into the bladder. In another embodiment,the agonist-containing composition described herein is miscible withwater due to the co-solvency of the penetration enhancer. An amphiphilicsolvent system, such as capsaicin dissolved in diethyleneglycolmonoethyl ether, may impart sufficient solubility of capsaicin in waterto allow direct instillation of the resulting monophasic mixture.

13.6 Prostatic Hyperplasia

Prostatic hyperplasia is a condition affliction many millions of malesaround the world. Notably, hyperpoliferative prostate cells share manyfeatures of cancer cells (which, as described supra, apoptose inresponse to capsaicin). See Tayeb et al., 2003, Br J Cancer 88:928-32.This condition can be treated by administration of the compositions ofthe invention. In one embodiment, the composition is administered in theform of a microemulsion such as is described infra in Section 13instilled into the urethra, would be of particular value to treat (andperhaps reverse) prostatic hyperplasia. In addition to treatment ofhyperplasia, instillation of a composition of the invention should alsoreduce the discomfort of urination symptom characteristic of prostatichyperplasia. Injecting capsaicin directly into the prostate would affectthe pacemaker and hyperplastic cells that express TRPV1. See Exintariset al., 2002, J. Urol. 168:315-22.

13.7 Psoriasis, Dermatitis, Pruritis and Itch

The efficacy of capsaicin against psoriasis, dermatitis, pruritis anditch has been documented in well-controlled clinical trials (e.g., seeBernstein et al., 1986, “Effects of Topically Applied Capsaicin onModerate and Severe Psoriasis Vulgaris,” J Am Acad Dermatol 15:504-507;Ellis et al., 1993, “A Double-Blind Evaluation of Topical Capsaicin inPruritic Psoriasis,” J Am Acad Dermatol 29:438-42). Such treatmentrequires application of low concentration creams many times per day formany weeks. In accordance with the present invention, the compositionsdescribed herein are applied topically to sites of psoriasis,dermatitis, pruritis or itch. It is expected that this treatment willprovide superior and longer lasting relief than presently availablemethods.

13.8 Warts

The common wart, or Verruca vulgaris, occurs in between 5 percent and 10percent of children and in a smaller percentage of adults. The standardtreatment is freezing with drops of liquid nitrogen, or cryotherapy.This procedure can be effective, but it can require months of repeated,painful applications that are scary to many children and can sometimeslead to blisters and infections. Also, cryotherapy has been reported tobe of limited efficacy. The capsaicin-containing compositions of thisinvention find use in the treatment of common warts. It is expected thatthis treatment will provide superior and longer lasting relief thanpresently available methods.

13.9 Migraine and Headache

Migraine (including migraine with aura) and headache (e.g., clusterheadache) are characterized by disabling pain and hyperactivation of thetrigeminal nervous system. Evidence exists that topical application ofTRPV1 agonists onto nasal mucosa can prevent or reverse headache. See,e.g., Saper et al., 2002, Arch Neurol 59:990-4; and Vass et al., 2001,Neuroscience 103:189-201. As the trigeminal nervous system innervatesthat skin of the face and head, as well as the mucosa of the nasalcavity. According to the invention, topical application of thecompositions of the invention to trigeminal nervous system (e.g.,application to the forehead or other parts of the face or head or intonasal passages) is used to prevent or reduce the symptoms of headache.

13.10 Wrinkles

Many wrinkles are caused by tonic activation of muscles underlying theskin. A reflex arch involving sensory nervous system hyperactivity islikely to be involved. Application of compositions of the presentinvention is used to reduce the depth or extent of wrinkles, or preventthe formation of wrinkles.

13.11 Tinnitus

There are many similarities between the symptoms and signs of severetinnitus and chronic pain, e.g., some individuals with severe tinnitusperceive sounds to be unpleasant or painful and some of the same drugsare used to treat both conditions (Moller, 2000, “Similarities betweensevere tinnitus and chronic pain” J Am Acad Audiol. 11:115-24). Littleis known about the anatomic location of the changes that causestinnitus, but it may be the inferior colliculus, as well as otherstructures. TRPV1 are found in hair cells and supporting cells of theorgan of Corti and the spiral ganglion cells of the cochlea. Animalstudies indicate that the main action of capsaicin is on outer haircells and suggest that TRPV1 in the cochlea play a role in cochlearhomeostasis (Zheng et al., 2003, “Vanilloid receptors in hearing:altered cochlear sensitivity by vanilloids and expression of TRPV1 inthe organ of corti” J Neurophysiol. 90:444-55). Moreover, activation ofTRPV1 by endogenous ligands may contribute to hypersensitivity of theeighth nerve to hair cell inputs in a variety of pathologic conditions,such as tinnitus, Meniere's disease and migraine (Balaban et al., 2003,“Type 1 vanilloid receptor expression by mammalian inner ear ganglioncells” Hear Res. 175:165-70). It is expected that compositions of thepresent invention may be effective for treatment of tinnitus.

14. Screening Methods

In one aspect, the invention provides a method for identifying acomposition as useful for therapeutic delivery of a TRPV1 agonist to asubject by determining the depot effect for a solution consisting of thecomposition and the TRPV1 agonist or a different TRPV1 agonist, where adepot effect less than 0.25 indicates composition is useful fortherapeutic delivery of a TRPV1 agonist. In alternative embodiments, adepot effect less than 0.2, 0.1, 0.05, 0.01, 0.005 or 0.001 indicatescomposition is useful for therapeutic delivery of a TRPV1 agonist. Inone embodiment, there is a further step of determining the amount ofagonist delivered to skin epidermis and dermis after a specified timewhen the composition is applied to the skin surface.

In a related aspect, the invention provides a method for ranking two ormore compositions according to their utility for therapeutic delivery ofa TRPV1 agonist to a subject by determining for each composition thedepot effect for a solution consisting of the composition and the TRPV1agonist or a different TRPV1 agonist, comparing the values obtained foreach composition, and ranking them compositions according to the values,where a composition with a lower value is ranked more suitable fortherapeutic delivery of the TRPV1 agonist. In one embodiment, there is afurther step of determining, for each composition, the amount of agonistdelivered to skin epidermis and dermis after a specified time when thecomposition is applied to the skin surface, where a composition with ahigher value is ranked more suitable for therapeutic delivery of theTRPV1 agonist.

In various embodiments of these methods: (i) the composition containsone or more penetration enhancers; (ii) the composition is a compositionof the present invention; (iii) the TRPV1 agonist is capsaicin; (iv) thedepot effect is determined in vitro; (v) the depot effect is determinedusing mouse skin; or (v) the depot effect is determined using mouse skinassay of the present invention.

15. Preparation and Administration of Microemulsion

Compositions of the invention can be administered in the form of amicroemulsion. Methods for making microemulsions are generally known inthe art. See, e.g., Prince, 1970 “Microemulsions” J. Soc. Cosmet. Chem.21:193-204; Prince L. M. in MICROEMULSIONS—THEORY AND PRACTICE,Academic, New York 1977; Belloq A. M. et al; Adv Colloid Interface Sci20: 167, 1984 and Bourrel M., Schechter R. S. in MICROEMULSIONS ANDRELATED SYSTEMS, Dekker, New York, 1988.

In one version, a microemulsion comprising three components is prepared.The three components are an internal phase, an external phase, and oneor more emulsifiers.

The Internal Phase

The content of the internal phase depends on the nature of theTRPV1-agonist-containing composition used to make the microemulsion.When the composition is not miscible in water, the composition serves asthe internal phase (although it can be optionally combined with an oilin which the composition is miscible). When the composition (i.e.,comprising a TRPV1-agonist and a solvent system, e.g., 5% (w/v)capsaicin in diethylene glycol monoethyl ether) is either amphiphilic orhydrophilic, the composition is mixed with an oil in which it ismiscible. Examples of such oils include, for illustration and notlimitation, mineral oil, mink oil, linseed oil, tung oil, pine oil, andvegetable oils. The internal phase is dispersed as microdroplets (e.g.,having a diameter of about 10 to about 200 nm, usually about 10 to about60 nm).

The External Phase

The external phase is an aqueous liquid, such as water, saline, buffer,or the like. The external phase is the medium in which the microdropletsare dispersed.

The Emulsifier(s)

Examples of suitable emulsifiers (for illustration and not limitation)include mineral oil, Pluronic (BASF), polyethoxylated fatty acids, PEGdiester fatty acids, PEG fatty acid mixtures of mono- and di-esters,polyglycerized fatty acids, mono- and diglycerides, sterols and sterolderivatives, sugar esters, polyoxyethylene-polyoxypropylene blockcopolymers, sorbitan fatty acid esters, lower alcohol fatty acid esters,ionic surfactants, and non-ionic surfactants, and combinations thereof.

Optional Components

In addition, the microemulsion may include other components such asstability enhancing components (e.g., antimicrobials, antioxidants, andthe like) pH adjusting agents and the like, stabilizing agents, and thelike). However, usually these components are present in small amounts(e.g., less than about 5% (w/v), most often less than about 1%).

Preparation of Microemulsion

A microemulsion may be prepared by adding at least one emulsifier (e.g.polyglyceryl-6 dioleate) to the internal phase (e.g., 5% capsaicinsolution in diethylene glycol monoethyl ether mixed with oil). Themixture is then added to an external phase (e.g., water). The resultingmixture is vigorously stirred, sonicated or otherwise agitated with orwithout the application of heat until stable microdroplets are formedand a homogeneous distribution results. The pH can be adjusted toimprove the stability and/or physiological tolerance of themicroemulsion.

Concentrated formulations of a TRPV1 agonist can be prepared containingco-solvents, surfactants, emulsifiers, and thickeners in concentrationshigher than the expected final administered composition. (See Example6.) These formulations are “preconcentrates,” not intended for directadministration but require further dilution in a suitable vehicle toobtain the desired administration concentration.

Use of Microemulsion

The microemulsion is contacted with a tissue to which the TRPV1 agonistis intended to be delivered. Without intending to be bound by aparticular mechanism, it is believed that the microdroplets will fuserapidly with the target tissue (i.e., tissue with which themicroemulsion is in contact) and deliver the TRPV1 agonist and any othertherapeutically active agents to the tissue.

Examples of treatments for which microemulsion administration is usefulinclude, without limitation, (1) treatment of peripheral neuropathy,arthritis, psoriasis and frostbite (administration via bath); (2)treatment of neurogenic bladder, interstitial cystitis or similarconditions (instillation into the bladder); (3) treatment of prostatecancer or prostatic hyperplasia (instillation into the urethra); and (4)instillation into surgical wounds to treat or prevent pain.

The method of contacting of the tissue and microemulsion will varydepending on the nature of the tissue and condition to be treated. Oneexample of a method for administration is via a water-bath type device(i.e., a container of the microemulsion in which a target tissue, suchas a hand or foot, can be immersed).

For example, the present invention provides methods and devices foradministering a TRPV1 agonist composition (either a microemulsion oranother other TRPV1 agonist-containing composition, such as thosedescribed hereinabove) using a bath type device. In one version, thebath device includes a basin containing the TRPV1 agonist microemulsionand for receiving an affected area of tissue. For example, a hand, foot,elbow, or any other affected area may be immersed into the bath fortreatment. This type of device has certain advantages.

First, a bath type device provides a convenient way to help ensure thatthe entirety of the affected area is treated, because entire body partsmay easily be immersed into the bath for treatment. A therapeutic bathis especially suitable for patients suffering from certain types ofneuropathy, such as, diabetic peripheral neuropathy, which typicallyaffects and produces pain within the lower extremities first, i.e., thefeet. A therapeutic bath can also be used for the treatment ofmusculoskeletal pain. Patients suffering from diabetic peripheralneuropathy can immerse their feet into the bath device described hereinfor treatment using the methods described herein. Similarly, neuropathicpain within the upper extremities typically starts within thefingertips, before moving up the hands and arms. Therefore, atherapeutic bath is also especially useful for the treatment of thefingers and hands.

Second, use of a bath type device is helpful in controlling andenhancing drug delivery. This is because the environmental conditionsaffecting drug delivery can be closely regulated and modified. Forexample, temperature, hydration, salt content, and drug concentrationhave all been shown to have an affect on absorption of a drug throughthe skin. Therefore, regulation of these properties can help control orenhance drug delivery.

In its simplest form, the bath device provides a container to house atreatment fluid. The container is of sufficient size such that anaffected area may be immersed therein. The device may, for example, takethe form of a bath for the feet. However, it should be understood thatthe bath can be of any number of shapes and sizes, and therefore besuitable for immersion of any number of affected body parts. In oneembodiment, the bath comprises a basin for containing the TRPV1 agonistmicroemulsion and receiving a body part therein. The basin has a bottomsurface and a wall structure extending upwardly therefrom.

When the bath is a foot-bath type of device, the basin has a length andwidth sufficient to accommodate the feet of an average sized adult user.Similarly, the basin size can be selected so as to provide enough spaceto permit the user to easily insert and remove the feet therefrom. Inaddition, the bath may include certain anatomically designed features.For example the bath device can include separate foot rests, optionalsupports for the arches, or other contoured shapes designed for thefeet. The bath can be made of any appropriate material.

In some embodiments, the bath includes heating or cooling elementsdesigned to regulate the temperature of the basin or the microemulsioncontained therein. Increased temperatures may help facilitatepenetration of the TRPV1 agonist through the skin. Conversely, decreasedtemperatures may help improve tolerability to any discomfort resultingfrom exposure to certain TRPV1 agonists. The heating or cooling elementscan be electrically controlled or can be battery operated. In someembodiments, the heating element provides the capability of focusingheat on a specific region of the foot of the user, for example, by usinginfrared rays. However, any type of suitable heating element may beused. The bath may also include an ultrasound or a sonication element toemit waves through the TRPV1 agonist microemulsion. Without wishing tobe bound by any particular theory, it is thought that the use of suchwaves helps facilitate penetration of the TRPV1 agonist through theskin.

The bath may also have a lid or seal to entrap fumes and preventaccidental spillage. The lid may be entirely removable, or it may notbe. For example, the lid can be configured to snap fit over the basinsurface, or may be attached to the basin using any other suitableattachment device (e.g., hinges). In some embodiments, the lid isdesigned to cover the entire basin surface and form a seal around theaffected area while the affected area is receiving treatment.

A control panel may be used to control the heating, cooling, andsonication elements. The control panel may be powered by any suitablepower supply, for example, a power cord that plugs into a 110 V ACoutlet, or even a battery. Having the bath powered by a battery helpsfacilitate the portability of the device. In addition, the control panelmay have a timer device to keep track of treatment time and to notifythe user when the treatment is complete. Any number of additionalmodifications or controls may be added to the control panel as desired.

As noted above, one aspect of the invention provides a method ofadministering a TRPV1 agonist microemulsion using a bath device. Inoperation, the TRPV1 agonist microemulsion is added to the basin of thedevice. Then the affected body part is immersed within the microemulsionfor a predetermined period of treatment time. Treatment times will varydepending upon the chosen agonist. In one embodiment, capsaicin is theVR1 receptor antagonist. In one embodiment, the capsaicin can be addedto the basin as an microemulsion (e.g., as described above), or thecapsaicin microemulsion may be formed in the bath itself (e.g., byproviding the capsaicin in a suitable solvent system, adding anemulsifying agent, and then mixing with water).

In another aspect, the invention provides methods and devices foradministering the TRPV1 agonist in an article of clothing or garment.The garments may for example, be gloves, socks, or finger or toebooties, designed to be worn on the extremities of those affected byneuropathy. It is desirable that the garments are made of aclose-fitting and stretchable material that allows the microemulsion tobe coated thereon or impregnated therein. The material may be made ofany number of natural or synthetic fibers. The thickness and theelasticity of the garments will vary depending on the type ofmicroemulsion used and the type of garment desired. The garments may beof any length, and may be disposable or may be reusable. In someembodiments the garments are multi-layered. The layers can include anouter layer that is moisture and vapor impermeable.

16. Examples Example 1 Determination of TRPV1 Agonist Content Using theMouse Skin Absorption Assay and Demonstration of Rapid and EfficientDelivery of TRPV1 Agonists to Mammalian Skin

This example shows delivery of TRPV1 agonists using variouscompositions, and described the “mouse skin absorption assay.” The mouseskin absorption assay is an in vitro assay for measuring the deliveryand retention of a TRPV1 agonist. The assay is generally as described byKemppainen and Reifeinrath, 1990, in METHODS FOR SKIN ABSORPTION, CRCPublication (hereinafter, “Kemppainen”), with the followingmodifications: Tissue that was not used on the day of animal sacrificewas stored at less than −70° C., rather than at less than −60° C., untilthe day of the experiment; and ³H₂O with a specific activity of ˜0.5μCi/mL (rather than ˜0.3 μCi/mL) was used, and the receptor solution wascollected and analyzed at 30 minutes, rather than 20 minutes asdescribed in Kemppainen.

Skin Preparation

Mouse trunk skin (Nu/Nu) without obvious signs of skin disease, obtainedwithin 2 hours of death, was used in this study. When obtained it wascleared of subcutaneous tissue, sealed in a water-impermeable plasticbag, and, if not used on the day of arrival, stored at <−70° C. untilthe day of the experiment. Prior to use it was thawed by placing the bagin ˜37° C. water, and then rinsed in tap water to remove any adherentblood or other material from the surface.

Skin from a single donor was first cleared of all subcutaneous tissueand approximately 50% of the dermis by manual scalpel technique, andthen cut into multiple smaller sections large enough to fit onto 0.8 cm²Franz diffusion cells (Crown Glass Co., Somerville, N.J.). The dermalchamber (receptor side) was filled to capacity with a receptor solutionof phosphate-buffered isotonic saline (PBS), pH 7.4±0.1, and theepidermal chamber (donor side) was left open to the ambient laboratoryenvironment. The cells were then placed in a diffusion apparatus inwhich the dermal receptor solution is stirred magnetically at ˜600 RPMand which is maintained to achieve a skin surface temperature of33.0±1.0° C. Skin surface temperature from representative chambers weremeasured and recorded.

To assure the integrity of each skin section, its permeability totritiated water was determined before application of the test products(Franz et al., 1990, “The use of water permeability as a means ofvalidation for skin integrity in in vitro percutaneous absorptionstudies” Abst. J. Invest. Dermatol., 94:525). Following a brief (0.5 to1 hour) equilibrium period, ³H₂O (NEN, Boston, Mass., sp. Act. ˜0.5μCi/mL) was layered across the top of the skin by dropper so that theentire exposed surface was covered (approximately 100-150 μL). After 5minutes the ³H₂O aqueous layer was removed. At 30 minutes the receptorsolution was collected and analyzed for radioactive content by liquidscintillation counting, and skin integrity confirmed based onquantification of penetration. Typical skin specimens in whichabsorption of ³H₂O was less than 1.75 μL-equ were considered acceptable.

Prior to administration of the topical test formulations to the skinsections, the receptor solution was replaced with fresh 1:10 PBSsolution prior to dosing. Prior to dosing the dosing chamber half-cellwas removed to provide full access to the skin surface. All formulationswere then applied to the skin sections using a calibrated positivedisplacement pipette, with a target dosing volume of 10 μL/0.8 cm². Theformulation was spread throughout the surface of the skin using theTeflon tip of the pipette. One to five minutes after application thedonor half-cell of the Franz chamber was replaced to re-seal and tosecure the skin in the chamber.

At a pre-selected time after dosing (15 minutes) the receptor solutionwas removed in its entirety, and a 4 mL volume freeze dried (SavantSpeedVac) and saved for subsequent analysis.

Each skin section was surface washed with 0.5 mL of methanol twice toremove any residual TRPV1 agonist. Skin sections were removed from thechambered, scored along the circumference edge of the 0 ring indentationwith a scalpel, and the epidermis was gently teased from the dermis withfine-tipped forceps. The skin sections were then separated intoepidermis and dermis. Each separate epidermis and dermis was mixed with1 mL methanol and allowed to extract for approximately 24 hours at roomtemperature on a horizontal shaker.

Quantification of capsaicin was by High Performance LiquidChromatography with Mass Spectrometry detection (HPLC-MS). Briefly,HPLC-MS was conducted on a Hewlett-Packard 1100 Series HPLC system witha 1100 Series API-ES LC/MSD in positive ion mode. A solvent systemconsisting of 70% Acetonitrile+0.1% TEA, 30% water+0.1% Formic acid wasrun through a C18 Luna column (4.6×100 mm, 3 μm, Phenominex Inc.) at aflow rate of 0.5 mL/min (6.5 minute run duration). Twenty microliters ofsample were injected. Peak areas were quantified to concentration usingan external standard curve prepared using pure synthetic capsaicinstandard and quantified by external standard methods.

The results are summarized in Tables 4 and 5.

Example 2 Reduction of Nerve Fiber Functionality in Nude Mouse Skin

This example shows a reduction of nerve fiber functionality (NFF) asdemonstrated by PGP 9.5 immunostaining following application ofcapsaicin to mouse skin. The experiments were performed on 8-12 week oldnude mice (Nu/Nu) (Charles River). The mice were acclimated andarbitrarily divided into either 2 dose groups (for Experiment 1) of 12mice per group (6 male and 6 female) or 4 dose groups (for Experiment 2)of 20 mice per group (10 male and 10 female).

In Experiment 1, On Day 0, 15 μL of 10% (w/v) capsaicin in 100%diethylene glycol monoethyl ether (DGME) and 0.1% (w/v) ethyl cellulose(i.e., Formulation No. 23 containing 0.1% ethyl cellulose) was appliedto a 1 cm×1 cm area on the back of anesthetized mice of one group, whilethe control group received DGME dispersed in an adhesive matrix. InExperiment 2, On Day 0, 15 μL of either 15% (w/v) capsaicin in 15% oleicacid, 10% isopropyl myristate, 10% cetyl alcohol, 55% DGME and 10%methanol and 0.1% ethyl cellulose (i.e, Formulation No. 42 containing0.1% ethyl cellulose) or 15% capsaicin (w/v) in 20% oleyl alcohol and80% propylene glycol plus containing 0.1% ethyl cellulose (i.e.,Formulation No. 28 containing 0.1% ethyl cellulose) or 15% capsaicin(w/v) in 90% 1 menthone and 10% methanol plus 0.1% ethyl cellulose(i.e., Formulation No. 27 containing 0.1% ethyl cellulose) was appliedto a 1 cm×1 cm area on the back of anesthetized mice of three groups,while the control group did not receive any treatment. In bothexperiments, mice were maintained under general anesthesia during the30-minute treatment period and until the test articles were removed.After removal of the test article, the skin area was cleaned with acleansing gel (89.08% PEG 300, 1.0% Carbopol 1382™, 0.02% butylatedhydroxyanisole, 0.01% disodium edentate, pH 6.5). On Day 7, the micewere sacrificed and the tissue from the application site was collectedand split into two equivalent sections. One section of skin was placedon a piece of cardboard, fixed in 10% neutral buffered formalin andprocessed to a hematoxylin-eosin stained slide for evaluation ofinflammation and any other abnormalities. Another skin section wasprepared as a frozen block for immunohistochemistry with an antibody PGP9.5. This tissue was stained with an antibody for PGP 9.5 whichidentifies the skin sensory nerve fibers. Processed tissues wereevaluated by a board certified veterinary pathologist for (1) any andall lesions, with particular attention to inflammation and anymicrovascular changes, and (2) the presence, absence and anyabnormalities in the architecture of the skin sensory nerve fibers. Foreach specimen, nerve fiber density was determined by counting the numberof normal-appearing nerve fibers observed in at least four microscopicalfields (each circular microscopical field had a radius of 90 μm) andthen averaged. If the morphology of nerve fibers in a field was changed,i.e., there was swelling, blebbing or varicosities, and the majority ofnerve fibers appeared to exhibit morphological changes, then that fieldwas assigned a value of no normal nerve fibers. The reported data arethe decrease in average of number of nerve fibers per microscopicalfield per animal, compared to the respective controls. Average nervefiber densities between groups were compared in order to determine whatif any effects the various treatments had on nerve fiber density.

The results of the study in this example show that in Experiment 1, a10% capsaicin (w/v) in DGME and 0.1% ethyl cellulose (Form. No. 23)caused approximately a 49% decrease in nerve fiber density, compared toDGME dispersed in an adhesive matrix. This decrease is statisticallysignificant (p≤0.01). In Experiment 2, 15% capsaicin (w/v) in 15% oleicacid, 10% isopropyl myristate, 10% cetyl alcohol, 55% DGME, 10%methanol, and 0.1% ethyl cellulose (Form. No. 42) caused approximately a39% decrease in nerve fiber density, 15% capsaicin (w/v) in 20% oleylalcohol, 80% propylene glycol and 0.1% ethyl cellulose (Form. No. 28)caused approximately 70% decrease in nerve fiber density, while 15%capsaicin (w/v) in 90% 1 menthone, 10% methanol, and 0.1% ethylcellulose (Form. No. 27) caused approximately 75% decrease in nervefiber density, compared to the untreated control. All decreases in nervefiber density observed in this experiment were statistically significant(p≤0.01).

Example 3 Reduction of Nerve Fiber Functionality in Rat Vulva

This example shows the effect of local application of TRPV1 agonistformulations to rat vulva.

Sixty female retired breeder Sprague-Dawley rats were divided into 6groups. On day 0, rats were anesthetized with isoflurane anesthetic gasand then LMX5® anesthetic cream was applied to the entire vulvar area ofeach rat at least 30 minutes. The test formulations were applied using amicropipette to dispense 33 μL of each formulation. The formulationsapplied were diethylene glycol monoethyl ether (DGME) alone; 0.01%capsaicin (w/v) in DGME; 0.1% capsaicin (w/v) in DGME; 1%resiniferatoxin (w/v) in DGME; 3% capsaicin (w/v) in 90% (v/v) DGME and10% (v/v) DMSO, a 10% capsaicin (w/v) in DGME. The formulations wereleft on for 20 minutes with the exception of 10% capsaicin (w/v) in DGMEwhich was left on for 5 minutes. The remaining formulation was removedusing a cleansing gel (89.08% PEG 300, 1.0% Carbopol 1382™, 0.02%butylated hydroxyanisole, 0.01% disodium edentate, pH 6.5). The gel wasleft on for approximately 3-5 minutes and then removed using Kim wipesand genital swabs. On Day 7, the rats were sacrificed and a 1 mm punchbiopsy of the vulva was collected in ice cold Phosphate Buffered Salinefor preparation as a frozen block for immunohistochemistry. Frozensections made from this tissue were stained with anti-PGP 9.5 antibody,which stains the terminal sensory nerve fibers. The tissues wereevaluated by a board certified veterinary pathologist for (1) any andall lesions, with particular attention to inflammation and anymicrovascular changes, and (2) the presence, absence and anyabnormalities in the architecture of the terminal nerve sensory nervefibers. For each specimen, nerve fiber functionality was determined bycounting the number of nerve fibers observed in at least fourmicroscopical fields (each circular microscopical field has a radius of90 μm) and then averaged. If the morphology of nerve fibers in a fieldwas changed, i.e., there was swelling, blebbing or varicosities, and themajority of nerve fibers appeared to exhibit morphological changes, thenthat field was assigned a value of no normal nerve fibers. The reporteddata are the average of number of nerve fibers per microscopical fieldper animal. The findings between groups were compared in order todetermine what if any effects the various treatments have on nervefibers.

The results of this study are shown in FIG. 2. These results indicatedthat three capsaicin-containing formulations with capsaicinconcentrations greater than 0.01% (w/v) and higher caused astatistically significant (p≤0.1) decrease in nerve fiber density asdemonstrated by PGP 9.5 immunostaining following application periods of5 or 20 minutes. A 1% (w/v) liquid formulation of another TRPV1 agonistresiniferatoxin (RTX) when applied for 20 minutes produced a 28%decrease in nerve fiber density in this experiment; however, additionalexperiments are needed to establish statistical significance of theobserved decrease.

Example 4 Pain Behavior Following Treatment of Vulva

This example shows the amount of nocifensive behaviors produced byapplications of 10% capsaicin (w/v) in diethylene glycol monoethylether, 3% capsaicin (w/v) in diethylene glycol monoethyl ether,diethylene glycol monoethyl ether alone, and Zostrix®, a commerciallyavailable 0.075% (w/v) capsaicin cream formulation, when applied tovulva of retired Sprague-Dawley breeder rats.

Treatment Groups Application Duration of Dose Group Vol. (μL)Application (min.) N diethylene glycol 33 20 6 monoethyl ether (DGME)Zostrix ® 50 20 6  3% Capsaicin (w/v) in 33 20 6 100% DOME  10%Capsaicin (w/v) in 33 5 6 100% DOME

The rats were monitored for 5 minutes for a baseline pain behavior. Thetest article was applied. After the indicated application time, residualcapsaicin was removed from vulva with a cleansing gel (89.08% PEG-300,1.0% Carbopol 1382™, 0.02% butylated hydroxyanisole, 0.01% disodiumedentate, pH 6.5). Following application of the test article, painbehavior was monitored for 60 minutes. The following behaviors wereconsidered as pain responses: licking or grooming the vulva area,looking down at the vulva, sniffing the vulva, licking and chewingmotion with mouth, standing on hind legs and lifting tail, runningaround the cage, crawling along the bottom of the cage, sitting up andpointing nose towards abdomen, excessive or frantic grooming, stretchinghind legs, lifting tail, digging at the bottom of the cage, andstretching posture.

FIG. 3 shows the results of the experiment. Capsaicin containing (3% and10% w/v) liquid formulations and over-the-counter capsaicin containing(0.075%) cream (Zostrix®) produced greater pain response than diethyleneglycol monoethyl ether alone, when applied to rat vulva for either 5minutes or 20 minutes. However, there was no statistically significantdifference in pain responses among 3% w/v capsaicin liquid formulationand Zostrix®. Surprisingly, a 10% w/v capsaicin liquid formulationproduced statistically significant (p≤0.1) less pain response than theZostrix® cream containing 0.075% capsaicin.

Example 5 Pain Behavior Following Treatment of Skin

This example shows the amount of nocifensive behaviors in rats followinga single topical application of capsaicin (10% w/v) in diethylene glycolmonoethyl ether (Transcutol™) compared to a commercial concentrationcapsaicin cream (0.1% w/w; Capzasin-HP® capsaicin cream) or diethyleneglycol monoethyl ether alone.

Materials and Methods

Adult, male, Sprague-Dawley rats (250-300 g, N=19) were placed in achamber and anesthetized with 2-3% halothane in air. Once each ratexhibited a sufficient depth of anesthesia, as indicated by a lack ofwithdrawal response to pinching of the tail, the rat was removed fromthe chamber and fitted with a facemask delivering 1-2% halothane. Therat was placed on a warming blanket and positioned on its left side withboth hind limbs extended. The dorsum of the each hind paw was cleaned ofdebris and wiped with an isopropyl (rubbing) alcohol wipe. A chemicaldepilatory agent was applied to the dorsum of each hind paw. Ten-minuteslater, the depilatory agent was removed with gauze then the dorsum ofeach hind paw was wiped with an isopropyl alcohol wipe. The dorsum ofboth hind paws was inspected to ensure that no visible hair remained.The halothane was discontinued and rats were allowed to recover in theircages.

Once rats were ambulating normally, they were placed on an elevatedplastic mesh and covered with a plastic container. Rats were allowed toacclimate on the mesh for at least 30 minutes. Next, the number of timesthat a rat shook its right hind paw over a 5-minute period was recorded.A hind paw shake was recorded only if the rat clearly elevated its righthind paw and shook it while it was not ambulating. After this baselineevaluation, each rat was removed from the mesh and wrapped gently in acloth towel. The dorsum of the right hind paw was wiped with anisopropyl wipe. Immediately after the alcohol evaporated, one of thefollowing treatments was applied to the dorsum of the right hind paw.

i) Capzasin-HP® 0.1% capsaicin cream (˜0.15 g);

ii) 50 μl DGME containing 10% (w/v) capsaicin;

iii) 50 μl DGME.

A cotton-tipped applicator was used to apply the capsaicin cream and thecream was rubbed into the dorsum of the hind paw for 10 sec. Thecapsaicin liquid formulation and DGME were applied to the dorsum of theright hind paw with a pipetter and the rat was kept immobile for 10 sec.After application of each treatment, rats were placed back onto the meshand covered with the plastic container. The number of times that eachrat shook its right hind paw was recorded for consecutive 5-minuteperiods for a total of 90 minutes.

Statistical Analyses

The number of times that rats shook their right hind paw is presented asthe mean (±sem) for each treatment group. One-tailed t-tests were usedto determine if there were significant differences between treatmentgroups in the number of hind paw shakes during each 5 minute period andover the entire 90 minute period. For all analyses, a probability(P-value) of less than or equal to 0.05 was considered significant.

Results

During the baseline 5-minute period, rats did not shake their right hindpaw. As shown in FIG. 1, application of capsaicin (0.1%) cream evoked59.4±23.7 shakes over the 90-minute observation period. Application ofthe capsaicin liquid formulation (10% capsaicin (w/v) in DGME) producedsignificantly less hind paw shakes (10.2±2.4 shakes, P≤0.05). Incontrast, application of DGME alone produced almost no nocifensivebehavior (0.7±0.7 shakes) in the 90-minute period, which wassignificantly fewer hind paw shakes than that produced by 10% capsaicinin DGME (P≤0.01).

Discussion

The results of this example suggest that topical application of the 10%capsaicin liquid formulation produced less nocifensive behavior (i.e.,hind paw shakes) over a 90-minute observation period than did topicalapplication of a commercially available over-the-counterlow-concentration (0.1%) capsaicin cream. Application of the capsaicinliquid formulation did evoke more hind paw shakes than did diethyleneglycol monoethyl ether alone.

Example 6 Preparation of Microemulsion Containing 0.1% Capsaicin (w/v)

A microemulsion is produced by preparing a first composition containingcapsaicin and diethylene glycol monoethyl ether. The first compositionis made by dissolving 10 g of capsaicin in 100 mL diethylene glycolmonoethyl ether (DGME) to form the internal phase. A microemulsion isprepared by adding the first composition to 1 L (one liter) of mineraloil, followed by addition of 10 to 30% (w/w) caprylocaproyl macrogol-8glycerides (emulsifier), stirring until the caprylocaproyl macrogol-8glycerides are dissolved, to produce a second composition (“oil phase”).

Prior to use or administration, 100 mL of the oil phase is added to 1 Lof water or saline (external phase), followed by thorough mixing until astable microemulsion is formed. The resulting microemulsion contains0.1% capsaicin (w/v).

Example 7 Preparation of an Oil in Water Microemulsion

The following example shows an oil in water microemulsion formulation.

Excipient % w/w Tetrahydrofurfuryl alcohol 40 (Glycofurol ®) Vitamin ETPGS 20 Propylene glycol 10 Isopropyl myristate 10 Span 80 5 Tween 80 5Deionised water 10

All the excipients were weighed into a vial and heated on a water bathat 60° C. until a uniform solution was formed. The solution was allowedto cool down to room temperature. A cloudy dispersion of o/w emulsionwas obtained. In order to incorporate the drug into the formulation,capsaicin was weighed along with the rest of the excipients and heatedon the water bath and allowed to cool down to form an o/w emulsion.

Example 8 Preparation of a Liposome Microemulsion

This example shows a liposome microemulsion formulation.

Excipient % w/w Lipoid S-100-3 9 Lipoid S-PG-3 1 Propylene Glycol 65 PBS25

All the ingredients were weighed directly into a vial and heated on awater bath at 70° C. until all the lipids were solubilized. Toincorporate capsaicin, the drug was be weighed along with the mainexcipients of the formulation and heated on the water bath until auniform single phased solution was obtained. This solution was thenhomogenised until it naturally cooled down to the room temperature. Asmooth white cream was obtained. Formation of liposomes was checkedunder the microscope. A 1 in 10 and a 1 in 100 dilution of the liposomeformulation in deionised water did not alter the physicalcharacteristics of the liposomes as observed under a microscope.

Example 9 Preparation of a Cosolvent Composition

This example shows a co-solvent composition.

The co-solvent pre-concentrates essentially consist of a surfactant(Poloxamer) solubilized in alcohol. The following example of the solventsystems was formulated to contain 10% w/v concentration of capsaicin. Adilution of the formulation in an aqueous medium resulted in a clearsolution.

In order to incorporate the drug into the formulation, the desiredamount of capsaicin was weighed into the vial followed by the additionof ethanol to solubilize the drug. This was followed by the addition ofPoloxamer 407, which was solubilized, and the rest of the ingredientswere weighed into the bottle directly. The formulation was mixed on avortex mixer to form a clear liquid.

Co-solvent pre-concentrate Excipient % w/w Poloxamer 407 20 Ethanol 40Tetrahydrofurfuryl alcohol 5 (Glycofurol ®) Deionised Water 35

Although the foregoing invention has been described in some detail byway of illustration and examples for purposes of clarity ofunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications may be practiced without departingfrom the spirit and scope of the invention. All publications, patentsand patent applications cited herein are hereby incorporated byreference in their entirety for all purposes to the same extent as ifeach individual publication, patent or patent application werespecifically and individually indicated to be so incorporated byreference.

TABLE 1 Penetration Enhancer Chemical Class Diethylene glycol monoethylether Ether Benzyl alcohol Alcohol Isopropyl myristate Fatty acid ester1-Menthone Terpene (ketone) Dimethyl isosorbide Ureas Caprylic alcoholFatty Alcohol Lauryl alcohol Fatty Alcohol Oleyl alcohol Fatty AlcoholEthylene glycol Polyol Diethylene glycol Polyol Triethylene glycolPolyol Butylene glycol Polyol Valerie acid Fatty acid Pelargonic acidFatty acid Caproic acid Fatty acid (linear) Caprylic acid Fatty acid(linear) Lauric acid Fatty acid (linear) Oleic acid Fatty acid (linear)Isovaleric acid Fatty acid (branched) Methyl nonenoic acid Fatty acid(branched) Isopropyl butyrate Fatty acid ester Isopropyl hexanoate Fattyacid ester Butyl acetate Fatty acid ester Methyl acetate Fatty acidester Methyl valerate Fatty acid ester Ethyl oleate Fatty acid esterPoloxamer Surfactant d-Piperitone Terpene (ketone) d-Pulegone Terpene(ketone) Dimethylsolfoxide Sulfoxides n-Hexane Alkanes Citric acidOrganic acid

TABLE 2 Penetration Enhancer Chemical Class Ethanol Alcohol PropanolAlcohol Isopropanol Alcohol Ethyl acetate Ester Methyl propionate Fattyacid ester Methanol Alcohol Butanol Alcohol Tert-butanol Alcohol OctanolAlcohol

TABLE 3 Penetration Enhancer Chemical Class Myristyl alcohol FattyAlcohol Methyl nonenoyl alcohol Fatty Alcohol Cetyl alcohol FattyAlcohol Cetearyl alcohol Fatty Alcohol Stearyl alcohol Fatty AlcoholMyristic acid Fatty Acid Stearic acid Fatty Acid Isopropyl palmitateFatty acid ester Sodium lauryl sulfate Surfactant (anionic)Benzallconium chloride Surfactant (cationic) Brij 35 Surfactant(nonionic) Tween 80 Surfactant (nonionic) Citric acid Organic AcidSalicylic acid Organic Acid

TABLE 4 Agonist Form. Conc.* Component 1 Component 2 Component 3Component Component No. (% w/v) (v/v %) (v/v %) (v/v %) 4 (v/v %) 5 (v/v%) 1 15 1,3-Butanediol Oleic acid Benzyl alcohol — — (50) (40) (10) 2 15Diethylene glycol Oleic acid Benzyl alcohol — — mono ethyl ether (40)(10) (50) 3 15 Diethylene glycol Ethyl oleate Oleic Acid Isopropyl monoethyl ether (30) (10) myristate — (50) (10) 4 15 1,3-Butanedioln-Caproic acid Myristic acid Diethylene (50) (20) (10) glycol mono —ethyl ether (20) 5 15 Diethylene glycol Diethylene 1,3-Butanediol Benzyl— (60) glycol mono (15) alcohol ethyl ether (10) (15) 6 15 d-PiperitoneDiethylene Oleic Acid Ethyl oleate — (30) glycol mono (20) ethyl ether(20) (30) 7 15 d-Pipertone Benzyl alcohol Oleic acid Ethyl oleate — (30)(20) (20) (30) 8 15 I-Menthone Benzyl alcohol Oleic acid Ethyl oleate —(30) (20) (20) (30) 9 15 Diethylene glycol Phosphate Benzyl alcohol — —mono ethyl ether buffer (10) (20) (70) 10 15 Diethylene glycol Oleicacid Benzyl alcohol Dimethyl mono ethyl ether (30) (10) isosorbide —(55) (5) 11 15 Isopropyl butyrate Diethylene Benzyl alcohol Cetyl — (65)glycol mono (10) alcohol ethyl ether (5) (20) 12 15 Diethylene glycolOleic acid Benzyl alcohol Sodium mono ethyl ether (40) (10) laurylsulfate — (49.05) (0.05) 13 15 Diethylene glycol Oleic acid — — — monoethyl ether (40) (60) 14 15 Ethylene glycol Diethylene 1,3-Butanediol —— (60) glycol mono (20) ethyl ether (20) 15 15 Oleyl alcohol IsopropylBenzyl alcohol I-Menthone — (65) butyrate (10) (5) (20) 16 15 Diethyleneglycol Oleic acid Benzyl alcohol — — mono ethyl ether (40) (10) (50) 1715 1,3-Butanediol Oleic acid — — — (60) (40) 18 15 1,3-Butanediol Benzylalcohol Phosphate — — (70) (20) buffer (10) 19 15 Isopropanol — — — —(100) 20 15 Methyl propionate — — — — (100) 21 15 DimethylacetamideBrij35 Methylnonenoic — — (5) (1) alcohol (94) 22 15 n-HexaneMethylnonenoic — — — (10) acid (90) 23 10 Diethylene glycol — — — — monoethyl ether (100) 24 1 Diethylene glycol — — — — mono ethyl ether (100)25 10 Oleic acid Isopropyl Cetyl alcohol Diethylene (15) myristate (5)glycol mono (5) ethyl ether (75) 26 10 Diethylene glycol DMSO Olvanilmono ethyl ether (10) (90) 27 15 or 2 Menthone Methanol — — — (90) (10)28 15 Oleyl alcohol Propylene glycol — — — (20) (80) 29 5.12 Oleylalcohol Propylene glycol — — — (20) (80) 30 5.12 1,3-Butanediol Oleicacid — — — (60) (40) 31 5.12 Menthone Methanol — — — (90) (10) 32 1Oleic acid Isopropyl Cetyl alcohol Diethylene (15) myristate (5) glycolmono (5) ethyl ether (75) 33 1 Diethylene glycol DMSO — — — mono ethylether (10) (90) 34 1 Diethylene glycol Propylene Phosphate mono ethylether Glycol Buffer (10) (5) (40) 35 1 Methanol — — — — (100) 36 1Diethylene glycol — — — — mono ethyl ether (100) 37 0.1 Diethyleneglycol — — — — mono ethyl ether (100) 38 0.3 Diethylene glycol — — — —mono ethyl ether (100) 39 1 Diethylene glycol — — — — mono ethyl ether(100) 40 3 Diethylene glycol — — — — mono ethyl ether (100) 41 10Diethylene glycol — — — — mono ethyl ether (100) 42 15 Oleic acidIsopropyl Cetyl alcohol Diethylene Methanol (15) myristate (10) glycolmono (10) (10) ethyl ether (55) *Capsacin, except where noted.

TABLE 5 P = Amount D = E = in Amount Amount S = receptor in in AmountForm. Capsaicin Application fluid dermis epidermis in skin No. % w/vTime (nmol) (nmol) (nmol) (nmol) P/S D/E 1 15 15 0.39 8.8 16.1 24.90.0157 0.546 2 15 15 0.28 13.26 21.19 34.45 0.0081 0.626 3 15 15 0.4110.03 22.46 32.49 0.0126 0.447 4 15 15 0.36 13.83 16.47 30.3 0.0119 0.845 15 15 0.04 3.45 6.48 9.93 0.004 0.533 6 15 15 0.08 9.44 22.38 31.830.0025 0.422 7 15 15 0.66 9.02 14.16 23.18 0.0285 0.637 8 15 15 0.117.69 21.01 28.7 0.0038 0.366 9 15 15 0.19 7.73 15.05 22.79 0.0083 0.51310 15 15 0.69 8.76 23.82 32.59 0.0212 0.368 11 15 15 0.45 16.04 25.9842.02 0.0107 0.617 12 15 15 0.28 12.03 13.24 25.27 0.0111 0.909 13 15 150.54 19.2 18.26 37.46 0.0144 1.052 14 15 15 1.41 12.16 16.89 29.060.0485 0.72 15 15 15 0.19 12.48 35.91 48.4 0.0039 0.348 16 15 15 0.759.47 19.73 29.21 0.0257 0.48 17 15 15 0.09 16.51 17.39 33.89 0.0027 0.9518 15 15 0.36 9.22 9.34 18.56 0.0194 0.987 19 15 15 0.88 11.24 19.1430.39 0.029 0.587 20 15 15 0.2 22.99 31.09 54.08 0.0037 0.74 21 15 155.11 33.55 33.01 66.56 0.0768 1.017 22 15 15 2.9 44.32 31.7 76.02 0.03811.398 23 10 30 3.61 14.75 9.57 24.31 0.1485 1.541 23 10 15 1.97 21.8714.63 36.49 0.054 1.495 23 10 5 1.73 11.27 6.11 17.37 0.0996 1.845 24 115 0.44 3.7 1.08 4.78 0.0921 3.411 25 10 10 1.23 12.73 9.8 22.52 0.05461.299 27 15 15 0.06 24.26 62.96 87.22 0.0007 0.385 27 15 2 0.11 6.3322.33 28.66 0.0038 0.283 28 15 15 0.1 29.87 47.42 77.29 0.0013 0.63 2815 2 1.31 6.61 13.21 19.83 0.0661 0.5 32 5.12 15 0.01 nd nd 6.53 0.0015— 35 5.12 15 0.01 nd nd 7.1 0.0014 — 36 5.12 15 0.01 nd nd 4.04 0.0025 —42 15 15 0.02 28.1 27.71 55.81 0.0004 1.014

TABLE 6 EXEMPLARY SOLVENT SYSTEMS (v/v)  1. Oleyl alcohol 100%  2.Methylnonenoyl alcohol 100%  3. Methylnonenoic acid 100%  4. Menthone95%, Methanol 5%  5. Menthone 85% Methanol 15%  6. Menthone 80% Methanol20%  7. Menthone 75% Methanol 25%  8. Menthone 70% Methanol 30%  9.Oleyl alcohol 10% Propylene glycol 90%  10. Oleyl alcohol 25% Propyleneglycol 75%  11. Oleyl alcohol 30% Propylene glycol 70%  12. Oleylalcohol 35% Propylene glycol 65%  13. Oleyl alcohol 40% Propylene glycol60%  14. Oleyl alcohol 45% Propylene glycol 55%  15. Oleyl alcohol 50%Propylene glycol 50%  16. Oleyl alcohol 60% Propylene glycol 40%  17.Oleyl alcohol 70% Propylene glycol 30%  18. Oleyl alcohol 80% Propyleneglycol 20%  19. Methylnonenoyl alcohol 95% 1-Menthone 5%  20.Methylnonenoyl alcohol 90% 1-Menthone 10%  21. Methylnonenoyl alcohol80% 1-Menthone 20%  22. Methylnonenoyl alcohol 60% 1-Menthone 40%  23.Methylnonenoyl alcohol 40% 1-Menthone 60%  24. Methylnonenoyl alcohol30% 1-Menthone 70%  25. Methylnonenoyl alcohol 20% 1-Menthone 80%  26.Methylnonenoyl alcohol 10% 1-Menthone 90%  27. Methylnonenoyl acid 5%1-Menthone 95%  28. Methylnonenoyl acid 95% 1-Menthone 5%  29.Methylnonenoic acid 90% 1-Menthone 10%  30. Methylnonenoic acid 80%1-Menthone 20%  31. Methylnonenoic acid 60% 1-Menthone 40%  32.Methylnonenoic acid 40% 1-Menthone 60%  33. Methylnonenoic acid 30%1-Menthone 70%  34. Methylnonenoic acid 20% 1-Menthone 80%  35.Methylnonenoic acid 10% 1-Menthone 90%  36. Methylnonenoic acid 5%1-Menthone 95%  37. Methylnonenoyl alcohol 95% oleic acid 5%  38.Methylnonenoyl alcohol 90% oleic acid 10%  39. Methylnonenoyl alcohol80% oleic acid 20%  40. Methylnonenoyl alcohol 70% oleic acid 30%  41.Methylnonenoyl alcohol 95% Dimethylacetamide 5%  42. Methylnonenoylalcohol 90% Dimethylacetamide 10%  43. Methylnonenoyl alcohol 80%Dimethylacetamide 20%  44. Methylnonenoyl alcohol 50% Methylnonenoicacid 50%  45. Methylnonenoyl alcohol 70% Methylnonenoic acid 30%  46.Methylnonenoyl alcohol 80% Methylnonenoic acid 20%  47. Methylnonenoylalcohol 90% Methylnonenoic acid 10%  48. Methylnonenoyl alcohol 40%Methylnonenoic acid 60%  49. Methylnonenoyl alcohol 30% Methylnonenoicacid 70%  50. Methylnonenoyl alcohol 20% Methylnonenoic acid 80%  51.Methylnonenoyl alcohol 10% Methylnonenoic acid 90%  52. Methylnonenoylalcohol 90% Oleyl alcohol 10%  53. Methylnonenoyl alcohol 80% Oleylalcohol 20%  54. Methylnonenoyl alcohol 70% Oleyl alcohol 30%  55.Methylnonenoyl alcohol 60% Oleyl alcohol 40%  56. Methylnonenoic alcohol50% Oleyl alcohol 50%  57. Methylnonenoyl alcohol 40% Oleyl alcohol 60% 58. Methylnonenoyl alcohol 30% Oleyl alcohol 70%  59. Methylnonenoylalcohol 20% Oleyl alcohol 80%  60. Methylnonenoyl alcohol 10% Oleylalcohol 90%  61. Methylnonenoyl alcohol 90% Propylene glycol10%  62.Methylnonenoyl alcohol 80% Propylene glycol20%  63. Methylnonenoylalcohol 70% Propylene glycol30%  64. Methylnonenoyl alcohol 60%Propylene glycol40%  65. Methylnonenoic alcohol 50% Propylene glycol50% 66. Methylnonenoyl alcohol 40% Propylene glycol60%  67. Methylnonenoylalcohol 30% Propylene glycol70%  68. Methylnonenoyl alcohol 20%Propylene glycol80%  69. Methylnonenoyl alcohol 10% Propylene glycol90% 70. Methylnonenoic acid 90% Oleyl alcohol 10%  71. Methylnonenoic acid80% Oleyl alcohol 20%  72. Methylnonenoic acid 70% Oleyl alcohol 30% 73. Methylnonenoic acid 60% Oleyl alcohol 40%  74. Methylnonenoic acid50% Oleyl alcohol 50%  75. Methylnonenoic acid 40% Oleyl alcohol 60% 76. Methylnonenoic acid 30% Oleyl alcohol 70%  77. Methylnonenoic acid20% Oleyl alcohol 80%  78. Methylnonenoic acid 10% Oleyl alcohol 90% 79. Methylnonenoic acid 90% Propylene glycol10%  80. Methylnonenoicacid 80% Propylene glycol20%  81. Methylnonenoic acid 70% Propyleneglycol30%  82. Methylnonenoic acid 60% Propylene glycol40%  83.Methylnonenoic acid 50% Propylene glycol50%  84. Methylnonenoic acid 40%Propylene glycol60%  85. Methylnonenoic acid 30% Propylene glycol70% 86. Methylnonenoic acid 20% Propylene glycol80%  87. Methylnonenoicacid 10% Propylene glycol90%  88. Methylnonenoyl alcohol 99% Brij35 1% 89. Methylnonenoyl alcohol 94.5% Dimethylacetamide 5% Brij35 0.5%  90.Methylnonenoyl alcohol 95.9% Dimethylacetamide 5% Brij35 0.1%  91. Oleicacid 15% Isopropyl myristate 15% DGME 60% Methanol 10%  92. Oleic acid20% Isopropyl myristate 15% DGME 55% Methanol 10%  93. Oleic acid 15%Isopropyl myristate 15% DGME 60% Cetyl alcohol 10%  94. Oleic acid 20%Isopropyl myristate 15% DGME 55% Cetyl alcohol10%  95. Oleic acid 10%Isopropyl myristate 10% Cetyl alcohol 10% DGME 60% Methanol 10%  96.Oleic acid 20% Isopropyl myristate 10% Cetyl alcohol 10% DGME 50%Methanol 10%  97. Oleic acid 30% Isopropyl myristate 10% Cetyl alcohol10% DGME 40% 'Methanol 10%  98. Oleic acid 40% Isopropyl myristate 10%Cetyl alcohol 10% DGME 30% Methanol 10%  99. Oleic acid 15% Isopropylmyristate 5% Cetyl alcohol 10% DGME 60% Methanol 10% 100. Oleic acid 15%Isopropyl myristate 10% Cetyl alcohol 5% DGME 60% Methanol 10% 101.Oleic acid 15% Isopropyl myristate 10% Cetyl alcohol 10% DGME 60%Methanol 5% 102. Oleic acid 10% Isopropyl myristate 10% Cetyl alcohol10% Methylnonenoyl alcohol 60% Methanol 10% 103. Oleic acid 20%Isopropyl myristate 10% Cetyl alcohol 10% Methylnonenoyl alcohol 50%Methanol 10% 104. Oleic acid 30% Isopropyl myristate 10% Cetyl alcohol10% Methylnonenoyl alcohol 40% Methanol 10% 105. Oleic acid 40%Isopropyl myristate 10% Cetyl alcohol 10% Methylnonenoyl alcohol 30%Methanol 10% 106. Oleic acid 15% Isopropyl myristate 5% Cetyl alcohol10% Methylnonenoyl alcohol 60% Methanol 10% 107. Oleic acid 15%Isopropyl myristate 10% Cetyl alcohol 5% Methylnonenoyl alcohol 60%Methanol 10% 108. Oleic acid 15% Isopropyl myristate 10% Cetyl alcohol10% Methylnonenoyl alcohol 60% Methanol 5% 109. Oleic acid 15% Isopropylmyristate 15% Methylnonenoyl alcohol 60% Methanol 10% 110. Oleic acid20% Isopropyl myristate 15% Methylnonenoyl alcohol 55% Methanol 10% 111.Oleic acid 15% Isopropyl myristate 15% Methylnonenoyl alcohol 60% Cetylalcohol 10% 112. Oleic acid 20% Isopropyl myristate 15% Methylnonenoylalcohol 55% Cetyl alcohol 10%

1-84. (canceled) 85: A pharmaceutical composition comprising atherapeutically effective amount of a TRPV1 agonist and one or morepenetration enhancers, and optionally one or more additionaltherapeutically active agents, wherein the composition delivers at leastabout 3 nmoles of agonist to skin as measured in a mouse skin absorptionassay, and wherein the pharmaceutical composition is in a form suitablefor administration to a subject.
 86. (canceled) 87: The pharmaceuticalcomposition of claim 85, comprising more than one penetration enhancer.88: The pharmaceutical composition of claim 85, wherein the compositioncomprises a solvent system comprising the one or more penetrationenhancers, and wherein the one or more penetration enhancers, takentogether, constitute at least about 20% (v/v) of the solvent system. 89:The pharmaceutical composition of claim 88, wherein the one or morepenetration enhancers, taken together, constitute at least about 75%(v/v) of the solvent system. 90: The pharmaceutical composition of claim89, wherein the one or more penetration enhancers, taken together,constitute at least about 95% (v/v) of the solvent system. 91.(canceled) 92: The pharmaceutical composition of claim 85, wherein thecomposition has a depot effect of less than about 0.25 as measured by amouse skin absorption assay. 93: The pharmaceutical composition of claim85, wherein the TRPV1 agonist is capsaicin. 94: The pharmaceuticalcomposition of claim 93, comprising 1% to 15% capsaicin. 95: Thepharmaceutical composition of claim 85, further comprising a localanesthetic.
 96. (canceled) 97: The pharmaceutical composition of claim85, wherein the composition is a microemulsion. 98-121. (canceled) 122:The pharmaceutical composition of claim 93, comprising greater than0.05% and less than 20% of capsaicin. 123: The pharmaceuticalcomposition of claim 85, wherein the composition delivers at least about16 nmoles of agonist to the skin. 124: The pharmaceutical composition ofclaim 123, wherein the composition delivers at least about 32 nmoles ofagonist to the skin. 125: The pharmaceutical composition of claim 124,wherein the composition delivers at least about 49 nmoles of agonist tothe skin. 126: The pharmaceutical composition of claim 125, wherein thecomposition delivers at least about 65 nmoles of agonist to the skin.127: The pharmaceutical composition of claim 85, wherein the compositiondelivers from about 3 nmoles to about 290 moles of agonist to the skin.128: The pharmaceutical composition of claim 88, wherein the one or morepenetration enhancers, taken together, constitute at least about 50%(v/v) of the solvent system. 129: The pharmaceutical composition ofclaim 88, wherein the one or more penetration enhancers is selected fromthe group consisting of ethers, esters, alcohols, fatty acids, fattyacid esters, fatty alcohols, polyols, terpenes, and amines. 130: Thepharmaceutical composition of claim 129, wherein the one or morepenetration enhancers is selected from the group consisting of fattyalcohols and terpenes. 131: The pharmaceutical composition of claim 88,wherein the one or more penetration enhancers is selected from the groupconsisting of l-menthone, dimethyl isosorbide, caprylic alcohol, laurylalcohol, oleyl alcohol, ethylene glycol, diethylene glycol, triethyleneglycol, butylene glycol, valeric acid, pelargonic acid, caproic acid,caprylic acid, lauric acid, oleic acid, isovaleric acid, isopropylbutyrate, isopropyl hexanoate, butyl acetate, methyl acetate, methylvalerate, ethyl oleate, poloxamer, d-piperitone, methylnonenoic acid,methylnonenoic alcohol, and d-pulegone.